Synthesis of a novel asparagine-linked heptasaccharide structure via p- methoxybenzyl-assisted β-mannosylation

Article abstract of DOI:10.1016/S0008-6215(00)00123-3

Synthesis of a core heptasaccharide asparagine N⁴-{α-D- mannopyranosyl-(1 → 6)-[(α-D-mannopyranosyl)-(1 → 3)]-[(2-acetamido-2- deoxy-β-D-glucopyranosyl)-(1 → 2)]-(β-D-mannopyranosyl)-(1 → 4)-(2- acetamido-2-deoxy-β-D-glucopyranosyl)-(1 → 4)-[(α-L-fucopyranosyl)-(1 → 6)]-2-acetamido-2-deoxy-β-D-glucopyranosyl}-L-asparagine (1a) found from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence of an extra GlcNAc residue linked to the 2-position of β-linked mannose. The synthesis was performed using p- methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected thiomannoside methyl 2-O-p-me thoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylidene-3-O- trimethylsilyl-1-thio-α-D-mannopyranoside was adopted for this particular purpose, which afforded β-mannoside p-methoxyphenyl 2,3-O-(p- methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylidene-β-D- mannopyranosyl-(1 → 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D- glucopyranoside stereoselectively in 75% yield. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00123-3

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Carbohydrate Research 328 (2000) 263–276
Synthesis of a novel asparagine-linked heptasaccharide
structure via p-methoxybenzyl-assisted b-mannosylation
Yuki Ohnishi, Hiromune Ando, Tsubasa Kawai 1, Yoshiaki Nakahara1, Yukishige Ito *
Institute of Physical and Chemical Research (RIKEN) and CREST,
Japan Science and Technology Corporation (JST), Wako-shi, Saitama 351-0198, Japan
Received 6 March 2000; accepted 17 April 2000
Abstract
Synthesis of a core heptasaccharide asparagine N4-{a-
[(2-acetamido-2-deoxy-b- -glucopyranosyl)-(1“2)]-(b- -mannopyranosyl)-(1“4)-(2-acetamido-2-deoxy-b-
pyranosyl)-(1“4)-[(a- -fucopyranosyl)-(1“6)]-2-acetamido-2-deoxy-b- -glucopyranosyl}- -asparagine (1a) found
D
-mannopyranosyl-(1“6)-[(a-
D
-mannopyranosyl)-(1“3)]-
D
D
D-gluco-
L
D
L
from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence
of an extra GlcNAc residue linked to the 2-position of b-linked mannose. The synthesis was performed using
p-methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected
thiomannoside methyl 2-O-p-methoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylidene-3-O-trimethylsilyl-1-thio-
a-
D
-mannopyranoside was adopted for this particular purpose, which afforded b-mannoside p-methoxyphenyl
-mannopyranosyl-(1“4)-3,6-di-O-
-glucopyranoside stereoselectively in 75% yield. © 2000 Elsevier Science Ltd. All
2,3-O-(p-methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylidene-b-
D
benzyl-2-deoxy-2-phthalimido-b-
rights reserved.
D
Keywords: Glycoprotein; Asparagine-linked glycans; Stereoselective; b-Manno glycoside; Intramolecular aglycon delivery
non-terminal modifications have been iden-
1. Introduction
tified. Particularly unique structures have been
identified by selection of lectin-resistant mu-
tant strains. For instance, Stanley and co-
workers recently reported the presence of
novel Asn-linked glycan chains that carry an
additional GlcNAc attached to a ManIII or
GlcNAcII residue (Fig. 1). Those structures
were found in dominant Chinese hamster
ovary (CHO) glycosylation mutant cells,
LEC14 [2] and LEC18 [3], which in turn were
selected by resistance to Pisum sati6um agglu-
tinin (PSA) and Lens culinaris agglutinin
(LCA), respectively.
Among various types of glycocojugate-re-
lated glycan chain structures those derived
from complex type asparagine (Asn) linked
glycoproteins are of particular interest in
terms of biological significance as well as
structural diversity and complexity [1]. Besides
the typical structural variations that arise
from branching, sialylation, fucosylation, bi-
secting GlcNAc addition and polylac-
tosaminylation, novel types of terminal and
* Corresponding author. Tel.: +81-48-4679430; fax: +81-
48-4624680.
E-mail address: yukito@postman.riken.go.jp (Y. Ito).
1 Present address: Department of Industrial Chemistry,
Tokai University, Hiratsuka-shi, Kanagawa 259-1292, Japan.
After extensive enzymatic trimming, a ma-
jor part of the LEC14-derived complex type
glycan chain was isolated as a nonasaccharide
1b [2], while the LEC18 N-glycan core struc-
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00123-3

264
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
ture was identified as the heptasaccharide 2b
[3]. In 1998 synthesis of the pentasaccharide
portion 2a, which is a partial structure of LEC
18-derived N-glycan 2b, was reported, where
b-linked ManIII was incorporated using con-
ventional insoluble silver salt protocol, which
proceeded in low yield (22%) and nonstereose-
lective (a:b=1.4:1) manner [4].
With recent establishment of p-methoxy-
benzyl (PMB)-assisted b-mannosylation as a
powerful method for the efficient and com-
pletely stereoselective synthesis of Asn-linked
glycans (Scheme 1) [5–8], we decided to apply
this methodology to the synthesis of the novel
heptasaccharide core structure 1a derived
from the LEC 14-derived glycan chain.
Fig. 1. Novel core structures of Asn-linked glycan chain from LEC14 (top) and LEC18 (bottom) CHO cells.
Scheme 1. PMB-assisted intramolecular aglycon delivery for stereoselective b-mannosylation.

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
265
Scheme 2. Synthesis of bMan-(1“4)-GlcNAc component. Reagents and conditions: (a) TIPDSCl2, imidazole, MeCN, 90%; (b)
p-MeOC6H4CH(OMe)2, PPTS, MeCN, 85%; (c) DIBAL, toluene, −40 °C, 86%; (d) Me3SiCl, imidazole, DMF, 94%; (e) DDQ,
,
,
CH2Cl2, 4A MS, 96%; (f) MeOTf, DBMP, Cl(CH2)2Cl, 4A MS, 78%.
Preparation of requisite 4 commenced with
2. Results and discussion
thiomannoside 5, which was reacted with
TIPDSCl2 and imidazole in acetonitrile to af-
ford 6 (90%). Subsequent formation of the
cyclic p-methoxybenzylidene acetal was ef-
fected by treatment with p-anisaldehyde
dimethylacetal and a catalytic amount of pyri-
dinium p-toluenesulfonate (PPTS) to afford 7
(85%). Reductive ring opening of the cyclic
acetal was effected by diisobutylaluminum hy-
dride (DIBAL-H) [9] in toluene at −40 °C,
which predominantly gave 2-O-PMB-pro-
tected 8a in 86% yield, together with a small
amount of regioisomer 8b (8a:8b=14:1). The
structure of 8a was confirmed by transforma-
tion into the corresponding 3-O-Ac derivative
The flexibility of our PMB-assisted b-man-
nosylation has been documented in the previ-
ous reports on the synthesis of typical core
structures of Asn-linked glycans [6,8]. Based
on this concept, we have successfully estab-
lished the highly optimized protocol to
achieve b-mannosylation, which affords bio-
logically
relevant
bMan-(1“4)-GlcNAc
building blocks in ꢀ75–85% yield [8]. Specifi-
cally, it was found that the efficacy of this
transformation is rather sensitive to the pro-
tecting group patterns and 4,6-O-cyclohexyli-
dene (3), and tetraisopropyldisiloxanylidene
(TIPDS) (4) protected mannosyl donors gave
optimum results. Since the first chemical syn-
thesis of prototypical complex type undecasac-
charide was recently achieved by using
4,6-O-cyclohexylidene-carrying 3 [8], the ap-
plicability of the alternative donor 4 for com-
plex oligosaccharide synthesis was explored in
this study.
1
8c. The H NMR spectrum of 8c revealed the
downfield shift of the H-3 proton (l 5.03).
The selectivity of ring opening was somewhat
sensitive to the temperature. Namely, the
8a:8b ratio dropped down to 7:1 and 2:1 at
−20 and 0 °C, respectively. Further protec-
tion of the C-3 position as a trimethylsilyl
ether afforded 4. b-Mannosylation was con-
ducted in a standard manner. Thus, treatment
of glucosamine derived acceptor 9 [10] with 4
The key disaccharide building block was
designed as 11 [7], which was prepared from
mannosyl donor 4 as depicted in Scheme 2.

266
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
attempted a-mannosylation of 2,3-diol 13 us-
and
2,3-dichloro-5,6-dicyanobenzoquinone
ing trichloroacetimidate 15 (BF3·OEt2,
CH2Cl2, −78 °C) was accompanied by the
migration of the TIDPS group, and formation
of the desired 3-O-glycosylated product was
not observed. In order to avoid these com-
plexities, 4,6-O-protection was exchanged to
the more robust di-tert-butylsilanediyl
(DTBS) group. Thus, treatment of 4,6-diol 12
with DTBS(OTf)2 and DBMP was followed
by the removal of the p-methoxybenzylidene
group with PPTS in methanol to give 16 in
93% yield. Selective mannosylation at the C-3
position with glycosyl chloride 14 by the ac-
tion of silver triflate proceeded uneventfully to
give trisaccharide 17 in 83% yield (Scheme 4).
The introduction of the crucial glucosamine
residue onto trisaccharide 17 was performed
by using glycosyl trichloroacetimidate 18 [15].
In spite of the presence of a 1,2-trans directing
group, trimethylsilyl trifluoromethansulfonate
(TMSOTf) catalyzed coupling yielded an
anomeric mixture of tetrasaccharides, which
was contaminated with silylated trisaccharide
21. This mixture was treated with PPTS (to
remove TMS) and separated by silica gel chro-
matography to afford the desired tetra-
saccharide 19 and corresponding a isomer 20
(ratio 5:1, combined yield 85%). Although
conditions for this glycosylation was examined
to some extent (Table 1), the formation of 20
could not be suppressed completely. At-
tempted glycosylation with fluoride 22
(AgOTf, Cp2HfCl2–CH2Cl2) [16] proved to be
less satisfactory (72%, 19:20=2.4:1). The fail-
ure to attain complete b-stereoselectivity indi-
cates that the combination of a highly
hindered 17 and the phthalimido-carrying gly-
cosyl donor may be a mismatched one [17].
Subsequent transformation to hexasaccha-
ride was performed as depicted in Scheme 5.
Previously reported azide 25 [6c] was em-
ployed as the fucosylated GlcNAc component.
Preparation of tetrasaccharide donor 24 from
18 was performed in a standard manner. The
fragment coupling reaction of these compo-
nents was performed by the action of
BF3·OEt2 in dichloromethane, and hexasac-
charide 26 was obtained in 38% yield. Activa-
tion with TMSOTf was less satisfactory to
afford a 24% yield of 26.
(DDQ) in dichloromethane in the presence of
,
,
4A molecular sieves (4A MS) afforded the
mixed acetal 10, which was isolated in 96%
yield after purification by florisil column chro-
1
matography. H NMR (270 MHz) analysis
revealed that it consisted of a single isomer,
and, based on the empirical rule reported pre-
viously [11], the configuration of the acetalic
carbon was assigned to be S. Low-field shifts
of H-1Man (l 5.94) and single benzylic proton
(l 5.11) were diagnostic for the stereochemical
assignment. Intramolecular aglycon delivery
(IAD) [12] was effected by methyl trifl-
uoromethanesulfonate (MeOTf) and 2,6-di-
tert-butyl-4-methylpyridine
(DBMP)
in
1,2-dichloroethane to afford a 78% yield of
b-manno glycoside 11. The 2,3-O-p-methoxy-
benzylidene cyclic acetal regenerated, pre-
sumably by the intramolecular trapping of the
immediate cationic product by trimethylsilyl
ether, again proved to be stereochemically ho-
mogeneous. The orientation of the p-
methoxyphenyl substituent was assigned as
1
endo, based on H NMR NOE experiments.
Compound 11 was selectively deprotected
to 12 and 13, which were subjected to a-man-
nosylation using either glycosyl chloride 14
[13] or trichloroacetimidate 15 [14]. (Scheme
3). Rather unexpectedly, reaction of 4,6-diol
12 with mannosyl chloride 14 turned out to be
non-regioselective to give a mixture of 4- and
6-O-glycosylated products. On the other hand,
Scheme 3. Selective deprotection of 11. Reagents and condi-
tions: (a) PPTS, MeOH–CH2Cl2, 85%; (b) TBAF, THF, 99%.

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
267
Scheme 4. Incorporation of (1“2)-linked bGlcNAc residue. Reagents and conditions: (a) DTBS(OTf)2, DBMP, DMF; (b) PPTS,
,
,
MeOH–CH2Cl2, 93% (two steps); (c) 14, AgOTf, DBMP, 4A MS 83%; (d) Lewis acid, solvent, 4A MS.
Table 1
Glycosylation of 17 with trichloroacetimidate 18
a
Entry
Solvent
Promoter (equiv)
Temperature (°C)
19/20 b
Yield (%)
1
2
3
4
5
6
7
CH2Cl2
toluene
1:1 toluene–hexane
toluene
toluene
TMSOTf (0.2)
TMSOTf (0.2)
TMSOTf (0.2)
TMSOTf (1.0)
BF3·OEt2 (0.2)
TESOTf (0.2)
TIPSOTf (0.2)
−20 to rt
0
0
3.3
6.7
4.0
5.0
1.4
5.2
5.8
B10
68
89
87
26
−20 to 0
0
0
0
toluene
toluene
56
44
a All reactions were performed in the presence molecular sieves.
1
b Determined by H NMR spectroscopy.
In order to incorporate an a-linked man-
nose residue, hexasaccharide 26 was subjected
to the removal of the DTBS group under
desilylating conditions. For this particular
purpose, tributylammonium hydrogen fluoride
(TBAHF), which has been reported by Fu-
rusawa and co-workers [18], gave us a
highly satisfactory result to afford 27 in an
excellent yield (90%). On the other hand, at-
tempted deprotection under more standard
In practice, regioselective glycosylation of 27
turned out to be less straightforward than ex-
pected. Silver triflate-promoted glycosylation
using glycosyl chloride 14 afforded C-6 manno-
sylated heptasaccaride 28 (34%), together with
the regioisomeric product (20%). This result
was in sharp contrast to our previous observa-
tion, where the glycosylation of pentasaccha-
ride 29 with 14 proceeded regioselectively to
afford the 6-O-glycosylated product in high
yield. Presumably, the steric hindrance of the
C-6 hydroxyl groups is comparable with that of
C-4 due to the presence of an axially oriented
glucosamine residue having bulky substituents.
conditions
using
tetrabutylammonium
fluoride–acetic acid was rather sluggish and
was accompanied by the formation of several
by-products.

268
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
Azide-carrying heptasaccharide 28 was con-
moisture were carried out under nitrogen or
argon atmosphere with anhydrous solvents.
Analytical thin-layer chromatography (TLC)
was developed on E. Merck Silica Gel 60 F256
plates (0.25 mm thickness). Preparative TLC
was performed using E. Merck Silica Gel 60
F256 plates (0.5 or 1.0 mm thickness). Column
chromatography was performed on E. Merck
Silica Gel 60 (60–230 mesh) or Florisil (60–
100 mesh, Kanto Chemical Co., Inc.). Flash
column chromatography was carried out using
E. Merck Silica Gel 60 (230–400 mesh). NMR
spectra were obtained on a JEOL EX-270,
EX-400, a-500, and/or b-600 spectrometer (1H
verted into glycosyl asparagine 32 as follows.
Removal of phthalimide groups with ethylene-
diamine in ethanol, followed by N-acetylation,
afforded 30. Chemoselective reduction of the
azide group with Lindlar catalyst, in the pres-
ence of Cbz-Asp-OBn-derived anhydride 31
[19], gave the protected asparagine-linked hep-
tasaccharide 32. Finally, hydrogenolytic de-
protection was performed with Pd(OH)2 –C in
ethanol–THF–water to afford the target Asn-
1
linked heptasaccharide 1a (Scheme 6). The H
NMR of 1a revealed the presence of seven
anomeric protons (a-Man×2, b-GlcNAc×3,
b-Man, and a-Fuc) with expected coupling
constants.
13
at 270, 400, 500, and/or 600 MHz, and C at
67.5, 100, 125, and/or 150 MHz) at ambient
temperature unless otherwise noted. 1H chemi-
cal shifts are in parts per million (l) adjusted
to Me4Si (CDCl3 and C6D6) at 0 ppm and
t-BuOH (D2O) at 1.23 ppm as an internal
reference, respectively. 13C chemical shifts
were referenced with CDCl3 at 77.0 ppm or
C6D6 at 128.0 ppm. Optical rotations were
measured on a JASCO DIP370 digital polar-
imeter. Mass spectra were obtained on a
JEOL SX-102A mass spectrometer in fast-
atom bombardment (FAB) mode with m-ni-
trobenzyl alcohol as the matrix with NaI as
the additive to promote molecular ions as
[M+Na]+.
In summary, the chemical synthesis of the
core heptasaccharide structure of LEC 14-
derived N-glycan was achieved using PMB-as-
sisted stereoselective b-mannosylation as the
key transformation.
3. Experimental
General methods.—Starting materials and
reagents were purchased from standard ven-
dors and used without purification unless oth-
erwise noted. All reactions sensitive to air or
Scheme 5. Coupling with fucosylated GlcNAc residue. Reagents and conditions: (a) CAN, MeCN–toluene–water, 71%; (b)
,
Cl3CCN, DBU, toluene, 86%; (c) BF3·OEt2, CH2Cl2, 4A MS, 38%.

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
269
,
Scheme 6. Synthesis of heptasaccharide asparagine. Reagents and conditions: (a) TBAHF, THF, 90%; (b) AgOTf, CH2Cl2, 4A
MS, 34%; (c) H2N(CH)2NH2, EtOH, reflux; (d) Ac2O, MeOH, 94%; (e) H2, Lindlar cat., EtOAc–MeOH, 63%; (f) H2,
Pd(OH)2–C, EtOH–THF–water, 61%.
H-4, H-6a), 3.91–3.82 (m, 3 H, H-3, H-5,
H-6b), 2.55 (d, 1 H, J 4.0 Hz, OH), 2.44 (d, 1
H, J 5.3 Hz, OH), 2.11 (s, 3 H, SMe), 1.12–
1.04 (m, 28 H, i-Pr). 13C NMR (67.5 MHz,
CDCl3): l 86.0, 72.9, 72.4, 72.3, 67.4, 61.0,
17.5, 17.20, 17.16, 17.0, 13.7, 13.6, 13.2, 12.5,
12.4. Anal. Calcd for C19H40O6SSi2: C, 50.40;
H, 8.90. Found: C, 50.66; H, 8.91.
Methyl
oxanylidene)-1-thio-h-
4,6-O-(1,1,3,3-tetraisopropyldisil-
-mannopyranoside (6).
D
—1,3 - Dichloro - 1,1,3,3 - tetraisopropyldisilo-
xane (5.46 mL, 17.0 mmol) was added to an
ice-cold water soln of 5 (3.00 g, 14.2 mmol)
and imidazole (3.87 g, 56.5 mmol) in dry
acetonitrile (60 mL). After being stirred at
0 °C for 30 min, the mixture was quenched
with MeOH and filtered through Celite. The
filtrate was coevaporated with toluene, and
the residue was purified by silica gel column
chromatography (3:17 EtOAc–hexane) to af-
ford compound 6 (5.50 g, 90%); [h]D +85.3°
Methyl 2,3-O-p-methoxybenzylidene-4,6-O-
(1,1,3,3-tetraisopropyl)disiloxanylidene-1-thio-
h- -mannopyranoside (7).—Pyridinium p-
D
toluenesulfonate (PPTS, 68 mg, 0.27 mmol)
was added to an ice-cold water soln of com-
pound 6 (1.19 g, 2.63 mmol) and 4-methoxy-
benzaldehyde dimethyl acetal (0.7 mL, 4
1
(c 1.46, CHCl3); H NMR (270 MHz, CDCl3):
l 5.26 (s, 1 H, H-1), 4.19–4.06 (m, 3 H, H-2,

270
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
(ca. 6%). The position of the p-methoxybenzyl
group was confirmed by acetylation of 8a
mmol) in dry acetonitrile (12.5 mL). After
being stirred at room temperature (rt) for 15
h, the mixture was diluted with EtOAc, and
quenched by aq NaHCO3. The organic layer
was washed successively with aq NaHCO3,
water, and brine, dried over Na2SO4, and was
evaporated in vacuo. The residue was purified
by silica gel column chromatography (1:99
EtOAc–toluene), followed by a column of
Bio-beads S-X4, to afford compound 7 (1.27
g, 85%) as a diastereomeric mixture (endo:exo
1.3:1; assignments based on differential NOE);
1
(Ac2O–pyridine, 40 °C, 18 h) to give 8c; H
NMR (400 MHz, CDCl3): l 5.29 (s, 1 H,
H-1), 5.03 (dd, 1 H, J 9.4 and 3.2 Hz, H-3),
4.50 (t, 1 H, J 9.4 Hz, H-4), 2.10 and 2.09 (2
s, 6 H, SMe, Ac).
Compound 8a: [h]D +21.3° (c 0.85,
1
CHCl3); H NMR (270 MHz, CDCl3): l 7.25
(d, 2 H, J 8.7 Hz, Ar), 6.86 (d, 2 H, J 8.7 Hz,
Ar), 5.41 (s, 1 H, H-1), 4.68 (d, 1 H, J 11.2
Hz, benzylic), 4.41 (d, 1 H, J 8.7 Hz), 4.17
(dd, 1 H, J 12.5 and 1.7 Hz, H-6a), 4.11 (dd,
1 H, J 9.2 and 8.9 Hz, H-4), 3.88–3.77 (m, 4
H, H-2, H-3, H-5, H-6b), 3.81 (s, 3 H, OMe),
2.23 (d, 1 H, J 9.2 Hz, OH), 2.10 (s, 3 H,
SMe), 1.10–0.99 (m, 28 H, i-Pr); 13C NMR
(67.5 MHz, CDCl3): l 129.7, 129.3, 113.8,
82.8, 79.7, 73.1, 71.8, 67.9, 61.1, 55.3, 17.5,
17.4, 17.34, 17.25, 17.0, 13.9, 13.6, 13.3, 12.40,
12.37. Anal. Calcd for C27H48O7SSi2: C, 56.61;
H, 8.44. Found: C, 56.50; H, 8.53.
1
[h]D +43.3° (c 1.31, CHCl3); H NMR (270
MHz, CDCl3) major isomer: l 7.41 (d, 2 H, J
8.6 Hz, Ar), 6.85 (d, 2 H, J 8.6 Hz, Ar), 5.87
(s, 1 H, CHAr), 5.70 (s, 1 H, H-1), 4.24–4.07
(m, 3 H), 3.96–3.84 (m, 3 H), 3.81 (s, 3 H,
OMe), 2.14 (s, 3 H, SMe), 1.12–0.84 (m, 28
H, i-Pr); minor isomer: l 7.34 (d, 2 H, J 8.6
Hz, Ar), 6.89 (d, 2 H, J 8.6 Hz, Ar), 6.05 (s, 1
H, CHAr), 5.58 (s, 1 H, H-1), 4.42 (dd, 1 H,
J 7.9 and 5.3 Hz, H-3), 4.22–4.15 (m, 3 H),
3.96–3.85 (m, 2 H), 3.78 (s, 3 H, OMe), 2.10
1
Corresponding regioisomer 8b: H NMR
13
(s, 3 H, SMe), 1.12–0.84 (m, 28 H, i-Pr); C
(400 MHz, CDCl3): l 7.26 (d, 2 H, J 8.5 Hz,
Ar), 6.87 (d, 2 H, J 8.3 Hz, Ar), 5.25 (s, 1 H,
H-1), 4.63 (d, 1 H, J 11.7 Hz, benzylic), 4.52
(d, 1 H, J 11.5 Hz, benzylic), 4.22 (t, 1 H, J
9.2 Hz, H-4), 4.18 (dd, 1 H, J 12.5 and 1.5 Hz,
H-6a), 3.98–3.80 (m, 6 H, H-2, H-5, H-6b,
OMe), 3.67 (dd, 1 H, J 9.2 and 3.2 Hz, H-3),
2.53 (s, 1 H, OH), 2.09 (s, 3 H, SMe), 1.08–
1.02 (m, 28 H, i-Pr).
NMR (67.5 MHz, CDCl3) major isomer: l
160.2, 129.6, 127.7, 113.5, 103.5, 82.0, 79.3,
78.2, 70.5, 69.3, 60.8, 55.3; minor isomer: l
160.2, 130.9, 127.3, 113.7, 102.5, 82.4, 79.7,
76.5, 70.2, 65.3, 60.9, 55.2; additional peaks l
17.3, 17.2, 17.12, 17.07, 16.9, 16.8, 13.8, 13.4,
13.3, 13.13, 12.4, 12.3, 12.2. Anal. Calcd for
C27H46O7SSi2: C, 56.81; H, 8.12. Found: C,
56.79; H, 8.18.
Methyl
(1,1,3,3 - tetraisopropyl)disiloxanylidene - 3 - O-
trimethylsilyl - 1 - thio - h - - mannopyranoside
2-O-p-methoxybenzyl-4,6-O-
Methyl
(1,1,3,3-tetraisopropyl)disiloxanylidene-1-thio-
h- -mannopyranoside (8a).—Diisobutylalu-
2-O-p-methoxybenzyl-4,6-O-
D
D
(4).—Chlorotrimethylsilane (0.25 mL, 2.0
mmol) was added to an ice-cold water soln of
compound 8a (741.3 mg, 1.29 mmol) and imi-
dazole (267.9 mg, 3.94 mmol) in dry DMF (18
mL). The mixture was stirred at rt for 2.5 h,
quenched with Et3N, and diluted with EtOAc.
The organic layer was washed successively
with aq NaHCO3 and brine, dried over
Na2SO4, and evaporated in vacuo. The residue
was purified by silica gel column chromatog-
raphy (1:19 EtOAc–hexane) to afford com-
pound 4 (780.7 mg, 94%); [h]D +49.3° (c
minum hydride (DIBAL-H; 0.95 M solution
in hexane, 3.60 mL, 3.42 mmol) was added to
a precooled (−40 °C) soln of compound 7
(607.7 mg, 1.06 mmol) in dry toluene (20 mL).
The mixture was stirred at the same tempera-
ture for 1 h, quenched by 0.5 M aq potassium
sodium tartarate (20 mL), and then stirred at
rt for 1 h. The clear reaction mixture was
diluted with EtOAc. The organic layer was
washed successively with water and brine,
dried over Na2SO4, and was evaporated in
vacuo. The residue was purified by silica gel
column chromatography (1:9–3:7 EtOAc–
hexane) to afford compound 8a (524.4 mg,
86%) and the corresponding regioisomer 8b
1
0.92, CHCl3); H NMR (270 MHz, CDCl3): l
7.26 (d, 2 H, J 8.4 Hz, Ar), 6.83 (d, 2 H, J 8.4
Hz, Ar), 5.25 (d, 1 H, J 1.1 Hz, H-1), 4.60 (s,
2 H, benzylic), 4.38 (t, 1 H, J 9.2 Hz, H-4),

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
271
quenched with Et3N (3.0 mL), diluted with
Et2O, and filtered through Celite. The filtrate
was washed successively with aq NaHCO3,
water, and brine, dried over Na2SO4, and
evaporated in vacuo. The residue was purified
by silica gel column chromatography (1:19
EtOAc–toluene) to afford compound 11
(658.6 mg, 78%); [h]D +35.1° (c 0.61, CHCl3);
1H NMR (270 MHz, CDCl3): l 7.80–6.67 (m,
22 H, Ar), 5.94 (s, 1 H, CHAr), 5.61 (d, 1 H,
J 8.6 Hz, H-1GlcN), 5.05 (d, 1 H, J 12.9 Hz,
benzylic), 4.90 (brs, 1 H, H-1Man), 4.78 (d, 1
H, J 12.0 Hz, benzylic), 4.70 (d, 1 H, J 12.9
Hz, benzylic), 3.72 (s, 3 H, OMe), 3.70 (s, 3 H,
OMe), 2.92 (brd, 1 H, J 9.9 Hz, H-3Man),
1.02–0.51 (m, 28 H, i-Pr); 13C NMR (67.5
MHz, CDCl3): l 160.0, 155.3, 150.9, 138.9,
137.8, 133.6, 129.8, 128.5, 128, 4, 128.2, 128.0,
127.8, 127.7, 126.8, 123.2, 118.7, 114.3, 113.3,
104.4, 99.2, 97.6, 80.1, 78.7, 77.5, 77.2, 77.1,
76.0, 74.7, 74.6, 73.6, 68.9, 68.0, 55.8, 55.5,
55.2, 17.4, 17.3, 17.2, 17.1, 17.0, 16.8, 13.1,
13.0, 12.6, 12.2. Anal. Calcd for C56H53NO14:
C, 69.77; H, 5.54; N, 1.45. Found: C, 69.57;
H, 5.62; N, 1.21.
4.16 (dd, 1 H, J 12.5 and 1.7 Hz, H-6a), 3.82
(m, 1 H, H-6b), 3.80 (s, 3 H, OMe), 3.73 (brd,
1 H, J 9.2 Hz, H-5), 3.65 (dd, 1 H, J 3.3 and
1.1 Hz, H-2), 2.10 (s, 3 H, SMe), 1.16–1.02
(m, 28 H, i-Pr), 0.11 (s, 9 H, SiMe3); 13C
NMR (67.5 MHz, CDCl3): l 158.9, 130.9,
128.9, 113.5, 84.7, 80.3, 74.4, 73.4, 72.4, 66.8,
61.2, 55.3, 17.9, 17.4, 17.3, 17.2, 17.11, 17.05,
13.8, 13.5, 13.4, 13.1, 12.0. Anal. Calcd for
C30H56O7SSi3: C, 55.86; H, 8.75. Found: C,
55.79; H, 8.82.
p-Methoxyphenyl 3,6-di-O-benzyl-2-deoxy-
2 - phthalimido - i -
D
- glucopyranoside - methyl
4%,6% - O - (1,1,3,3 - tetraisopropyl)disiloxanyli-
dene-3%-O-trimethylsilyl-1%-thio-h-
ranoside-2%,4-p-methoxybenzylidene
D-mannopy-
acetal
(10).—DDQ (219 mg, 0.96 mmol) was added
to an ice-cold water mixture of compounds 4
(504.2 mg, 0.78 mmol) and 9 (580.2 mg, 0.98
,
mmol) and 4A MS in dry CH2Cl2 (15 mL).
After being stirred at rt for 2 h, the mixture
was quenched with an aq soln of ascorbic acid
(0.7%)–citric acid (1.3%)–NaOH (0.9%) (10
mL). The resulting lemon-yellow mixture was
diluted with Et2O, and filtered through Celite.
The filtrate was washed successively with aq
NaHCO3 and brine, and the combined or-
ganic layers were dried over Na2SO4 and
evaporated in vacuo. The residue was purified
by florisil column chromatography (1:49
EtOAc–toluene) to afford mixed acetal 10
(929.4 mg, 96%); 1H NMR (270 MHz,
CDCl3): l 5.94 (s, 1 H, H-1Man), 5.81 (s, 1 H,
CHAr), 5.55 (d, 1 H, J 8.2 Hz, H-1GlcN), 5.11
(d, 1 H, J 12.5 Hz, benzylic), 3.81 (s, 3 H,
OMe), 3.69 (s, 3 H, OMe), 2.21 (s, 3 H, SMe),
1.01–0.80 (m, 28 H, i-Pr), 0.04 (s, 9 H,
SiMe3); HRFABMS: Calcd for C65H87-
NNaO5SSi3 [M+Na]+: 1260.5002. Found:
1260.4921.
p-Methoxyphenyl 2,3-O-p-methoxybenzyli-
dene - i -
D
- mannopyranosyl - (1“4) - 3,6-di-O-
-glucopyra-
benzyl-2-deoxy-2-phthalimido-i-
D
noside (12).—TBAF (1 M solution in THF,
1.8 mL, 1.8 mmol) was added to an ice-cold
water solution of 11 (642.3 mg, 0.57 mmol) in
THF (15 mL), and the solution was stirred at
0 °C for 45 min. The resulting mixture was
diluted with CHCl3 and washed with water
(×2). The organic layer was dried over
Na2SO4 and evaporated in vacuo. The residue
was purified by silica gel column chromatog-
raphy (3:17–3:2 EtOAc–toluene) to afford 12
(499.0 mg, 99%); [h]D +47.0° (c 0.45, CHCl3);
1H NMR (270 MHz, CDCl3): l 7.67–6.66 (m,
22 H, Ar), 5.90 (s, 1 H, benzylidene), 5.62 (d,
1 H, J 8.2 Hz, H-1GlcN), 4.95 (s, 1 H, H-1Man),
4.92 and 4.72 (d, 1 H each, J 12.0 Hz, ben-
zylic), 4.56–4.45 (m, 4 H), 4.25 (dd, 1 H, J 9.6
and 8.9 Hz, H-3GlcN), 4.10–4.02 (m, 2 H),
3.86–3.65 (m, 2 H), 3.72 and 3.70 (s, 3 H
each, OMe), 3.49 (m, 1 H), 3.21 (m, 1 H,
H-5GlcN); 13C NMR (67.5 MHz, CDCl3): l
160.3, 155.3, 150.7, 138.1, 137.6, 133.8, 131.5,
129.1, 128.4, 128.1, 128.0, 127.93, 127.87,
127.3, 127.1, 123.3, 118.7, 114.3, 113.6, 104.9,
p - Methoxyphenyl O - [2,3 - O - (p - methoxy-
benzylidene) - 4,6 - O - (1,1,3,3 - tetraisopropyl)-
disiloxanylidene-i-
D
-mannopyranosyl]-(1“4)-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-i-
D-
glucopyranoside (11).—To a stirred mixture of
acetal 10 (929.4 mg, 0.75 mmol), DBMP
,
(796.0 mg, 3.88 mmol), and 4A MS (3.5 g) in
dry (CH2Cl)2 (90 mL) was added to a soln of
MeOTf in (CH2Cl)2 (1 M, 3.0 mL, 3.0 mmol)
at 0 °C. After being stirred at rt for 0.5 h and
then at 60 °C for 14 h, the reaction was

272
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
Calcd for C49H59NNaO13Si [M+Na]+:
98.3 (C-1II), 97.8 (C-1I), 79.9, 78.2, 77.5,
76.8, 76.0, 75.4, 74.7, 73.8, 69.9, 67.9, 62.1,
55.6, 55.4, 55.3; HRFABMS: Calcd for
C49H49NNaO14 [M+Na]+: 898.3051. Found:
898.3042.
920.3653. Found: 920.3651.
p-Methoxyphenyl 3,4,6-tri-O-benzyl-h- -
D
mannopyranosyl - (1“3) - 4,6 - di - tert - butyl-
silanediyl - i - - mannopyranosyl - (1“4) - 3,6-
di-O-benzyl-2-deoxy-2-phthalimido-i- -glu-
D
D
p-Methoxyphenyl 4,6-O-di-tert-butylsilane-
copyranoside (17).—Compounds 16 (274.0
mg, 0.31 mmol) and 14 (229.7 mg, 0.45
diyl-i-
D
-mannopyranosyl-(1“4)-3,6-di-O-
-glucopyra-
benzyl-2-deoxy-2-phthalimido-i-
D
,
noside (16).—A solution of di-tert-butysi-
lanediyl ditriflate (0.14 mL, 0.37 mmol) was
added to an ice-cold water soln of compound
12 (265.7 mg, 0.31 mmol) and DBMP (202
mg, 0.98 mmol) in dry DMF (10 mL), and the
mixture was stirred at rt for 1.5 h. The reac-
tion was quenched with Tris (37.5 mg), and
stirred at rt for 10 min. The resulting mixture
was diluted with EtOAc, and washed succes-
sively with aq Na2CO3, water, and brine. The
organic layer was dried over Na2SO4 and
evaporated in vacuo. The residue was passed
through a pad of silica gel (1:4 EtOAc–tolu-
ene) to afford a crude mixture of the corre-
sponding silylene (ca. 294 mg), which was
dissolved in 5:2 CH2Cl2–MeOH (7 mL). A
solution of PPTS (0.05 M in MeOH, 1 mL,
0.05 mmol) was added, and the solution was
stirred at rt for 2.5 h. The resulting mixture
was diluted with EtOAc and washed with aq
NaHCO3 and brine, successively. The organic
layer was dried over MgSO4 and evaporated
in vacuo. The residue was subjected succes-
sively to a column of Bio-beads SX-4 (toluene)
and silica gel column chromatography (1:4–
1:1 EtOAc–toluene) to afford 16 (253.9 mg,
mmol), DBMP (161 mg, 0.74 mmol), and 4A
MS (1.0 g) were suspended in dry CH2Cl2 and
cooled down to −40 °C. Silver triflate (147.1
mg, 0.57 mmol) was added, and the mixture
was stirred for 2 h while gradually being
warmed up to −30 °C. The reaction was
quenched with aq Na2CO3, diluted with
EtOAc, and filtered through Celite. The
filtrate was dried over MgSO4 and the solvent
was evaporated in vacuo. The residue was
purified by silica gel column chromatography
(7:93–3:17 EtOAc–toluene) to afford com-
pound 17 (347.1 mg, 83%); [h]D +27.4 (c
1
0.57, CHCl3); H NMR (270 MHz, CDCl3): l
7.67–6.67 (33 H, Ar), 5.58 (d, 1 H, J 8.3 Hz,
H-1II), 5.52 (dd, 1 H, J 3.1 and 1.8 Hz, H-2IV),
5.21 (d, 1 H, J 1.8 Hz, H-1III), 4.89–4.29 (m,
13 H), 4.14–3.45 (m, 14 H), 3.70 (s, 3 H,
OMe), 3.12 (ddd, 1 H, J 9.9, 5.0, and 5.0 Hz,
H-5), 2.49 (s, 1 H, OH), 2.13 (s, 3 H, OAc),
1.03 (s, 9 H, t-Bu), 0.94 (s, 9 H, t-Bu); 13C
NMR (67.5 MHz, CDCl3): l 169.8, 155.4,
150.8, 138.3, 138.0, 137.9, 137.7, 133.8, 131.5,
128.5, 128.43, 128.36, 128.32, 128.2, 128.1,
127.9, 127.8, 127.73, 127.6, 127.60, 127.2,
123.3, 118.7, 114.3, 100.4 (C-1III), 99.7 (C-1IV),
97.7 (C-1II), 79.6, 78.5, 78.2, 75.1, 74.7, 74.6,
74.5, 73.51, 73.46, 73.3, 71.9, 71.8, 71.0, 70.8,
69.2, 68.5, 68.4, 66.2, 55.6, 55.5, 27.5, 26.9,
22.5, 21.0, 19.8; HRFABMS: Calcd for
1
93%); [h]D +47.5° (c 0.75, CHCl3); H NMR
(270 MHz, CDCl3): l 7.67–6.68 (18 H, Ar),
5.60 (d, 1 H, J 8.2 Hz, H-1GlcN), 4.79 (d, 1 H,
J 12.4 Hz, benzylic), 4.74 (d, 1 H, J 12.0 Hz,
benzylic), 4.72 (s, 1 H, H-1Man), 4.53–4.41 (m,
4 H), 4.14 (dd, 1 H, J 9.6 and 8.2 Hz, H-
3GlcN), 4.06 (dd, 1 H, J 10.1 and 5.1 Hz,
H-6aGlcN), 3.70 (s, 3 H, OMe), 3.33 (m, 1 H,
H-3Man), 3.14 (ddd, 1 H, J 9.7, 9.6, and 4.8
Hz, H-5GlcN), 2.63 (d, 1 H, J 4.5 Hz, OH), 2.57
(d, 1 H, J 2.1 Hz, OH), 1.01 (s, 9 H, t-Bu),
0.97 (s, 9 H, t-Bu); 13C NMR (67.5 MHz,
CDCl3): l 155.3, 150.6, 138.1, 137.5, 133.7,
128.4, 127.9, 127.8, 127.6, 123.3, 118.7, 114.3,
100.6 (C-1Man), 97.8 (C-1GlcN), 78.6, 77.7, 77.2,
74.8, 74.7, 74.1, 73.8, 73.7, 70.9, 70.3, 68.5,
66.0, 55.6, 27.5, 27.1, 22.7, 20.0; HRFABMS:
C78H89NNaO19Si
Found: 1394.5677.
[M+Na]+:
1394.5696.
p-Methoxyphenyl 2-O-acetyl-3,4,6-tri-O-
-mannopyranosyl-(1“3)-[3,4,6-tri-
O-benzyl-2-deoxy-2-phthalimido-i- -glucopy-
ranosyl-(1“2)]-4,6-di-tert-butylsilanediyl-i-
-mannopyranosyl-(1“4)-3,6-di-O-benzyl-
2 - deoxy - 2 - phthalimido - i - - glucopyrano-
benzyl-h-
D
D
D
D
side (19).—Trimethylsilyl trifluoromethansul-
fonate (29 mL, 0.16 mmol) was added to a
precooled (−20 °C) mixture of compound 17
(194.6 mg, 0.14 mmol) and 18 (252.2 mg, 0.43
,
mmol) in dry toluene (10 mL) containing 4A

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
273
added to a biphasic solution of 19 (263.3 mg,
0.14 mmol) in 4:3:2 toluene–MeCN–water
(3.6 mL), and the mixture was stirred at rt.
After 1 h, the mixture was diluted with
EtOAc, washed successively with water, aq
NaHCO3, and brine, dried over MgSO4, and
evaporated in vacuo. The residue was sub-
jected to a column of Bio-beads SX-4 (tolu-
ene), followed by silica gel column
chromatography (3:17–1:3 EtOAc–toluene)
to afford 23 (176.9 mg, 71%); [h]D +67.0° (c
MS (1.0 g), and the mixture was stirred at
0 °C for 1 h. The reaction was quenched with
aq NaHCO3, diluted with EtOAc, and filtered
through Celite. The filtrate was washed suc-
cessively with aq NaHCO3 and brine, dried
over MgSO4, and evaporated in vacuo. The
residue was subjected to a column of Bio-
beads SX-1 (toluene) that was followed by
further purification with silica gel column
chromatography (1:4–1:1 EtOAc–toluene) to
afford a mixture of 19/20 (256 mg), which
contains trimethylsilylated acceptor 21.
1
0.44, CHCl3); H NMR (270 MHz, C6D6): l
This mixture was treated with PPTS (18 mg,
0.072 mmol) in 3:5 MeOH–CH2Cl2 (8 mL).
After 12 h, the mixture was diluted with
EtOAc, and washed successively with aq
NaHCO3 and brine. The organic layer was
dried over MgSO4 and the solvent was evapo-
rated in vacuo. The residue was purified
by silica gel column chromatography (1:4–1:1
EtOAc–toluene) to afford a mixture of 19/20
(5:1 ratio, 233.3 mg, 85%) and trisaccharide 17
(17.3 mg, 9%). Pure 19 was finally obtained
by preparative TLC (5:4:1 toluene–CHCl3–
EtOAc).
7.93–6.69 (m, Ar), 6.03 (brs, 1 H, H-2IV), 5.87
(d, 1 H, J 8.4 Hz, H-1IX), 5.62 (s, 1 H, H-1IV),
5.47 (dd, 1 H, J 7.7 and 5.8 Hz, H-1II), 5.13
(d, 1 H, J 11.0 Hz, benzylic), 1.72 (s, 3 H, Ac),
1.33 (s, 9 H, t-Bu), 1.04 (s, 9 H, t-Bu); 13C
NMR (67.5 MHz, C6D6): l 104.6, 100.9, 98.6,
93.4, 82.6, 80.3, 80.0, 79.1, 78.7, 76.6, 75.9,
75.7, 75.5, 75.1, 74.7, 74.5, 74.3, 74.2, 73.9,
73.5, 72.8, 72.0, 71.6, 70.3, 70.0, 69.2, 66.5,
58.4, 57.3, 28.2, 27.5, 23.0, 20.7, 20.2; HR-
FABMS: Calcd for C106H114N2NaO24Si [M+
Na]+: 1849.7847. Found: 1849.7806.
2-O-Acetyl-3,4,6-tri-O-benzyl-h-
pyranosyl - (1“3) - [3,4,6 - tri - O - benzyl - 2 - de-
oxy-2-phthalimido-i- -glucopyranosyl-(1“2)]-
4,6-di-O-tert-butylsilanediyl-i- -mannopyra-
nosyl - (1“4) - 3,6 - di - O - benzyl - 2 - deoxy - 2-
phthalimido- -glucopyranosyl trichloroacetimi-
D
-manno-
Compound 19: [h]D +56.3° (c 0.82,
1
CHCl3); H NMR (270 MHz, C6D6): l 7.91–
D
6.56 (m, Ar), 6.13 (d, 1 H, J 8.6 Hz, H-1II),
6.04 (dd, 1 H, J 3.0 and 1.8 Hz, H-2IV), 5.90
(d, 1 H, J 11.0 Hz, benzylic), 3.21 (s, 3 H,
OMe), 1.72 (s, 3 H, OAc), 1.33 (s, 9 H, t-Bu),
1.05 (s, 9 H, t-Bu); 13C NMR (67.5 MHz,
CDCl3): l 169.4, 156.0, 151.4, 139.6, 139.3,
139.2, 139.1, 139.0, 138.9, 133.6, 133.4, 132.8,
132.0, 128.8, 128.7, 128.6, 128.54, 128.49,
128.23, 128.17, 128.1, 127.9, 127.8, 127.3,
127.2, 127.0, 126.7, 123.8, 123.5, 119.1, 114.9,
104.6 100.9 98.6, 98.2, 82.6, 80.4, 80.2, 79.0,
78.9, 78.8, 76.5, 75.9, 75.7, 75.5, 74.9, 74.8,
74.6, 74.2, 73.9, 73.7, 73.6, 72.8, 72.0, 71.7,
70.1, 69.9, 69.2, 66.5, 60.1, 57.3, 56.7, 55.1,
28.2, 27.5, 23.0, 20.7, 20.2, 14.4; HRFABMS:
Calcd for C113H120O25N2SiNa [M+Na]+:
1955.7847. Found: 1955.7817.
D
D
date (24).—DBU (1 M solution in toluene, 30
mL, 0.03 mmol) was added to an ice-cold
water solution of compound 23 (109.5 mg,
0.06 mmol) and trichloroacetonitrile (1.0 mL,
10 mmol) in dry toluene (3.5 mL), and the
mixture was warmed up to rt. After 4.5 h, the
mixture was subjected to silica gel column
chromatography (1:9 EtOAc–toluene) to af-
1
ford compound 24 (102.0 mg, 86%); H NMR
(270 MHz, C6D6): l 8.51 (s, 1 H, NH), 7.82–
6.70 (m, Ar), 6.05 (dd, 1 H, J 2.8 and 2.0 Hz,
H-2IV), 5.88 (d, 1 H, J 8.4 Hz, H-1II or H-1IX),
5.64 (s, 1 H, H-1IX or H-1II), 5.13 (d, 1 H, J
11.0 Hz, benzylic), 1.73 (s, 3 H, OAc), 1.32 (s,
9 H, t-Bu), 1.05 (s, 9 H, t-Bu).
2-O-Acetyl-3,4,6-tri-O-benzyl-h-
pyranosyl - (1“3) - [3,4,6 - tri - O - benzyl - 2 - de-
oxy-2-phthalimido-i- -glucopyranosyl-(1“2)]-
4,6-di-tert-butylsilanediyl-i- -mannopyran-
osyl-(1“4)-3,6-di-O-benzyl-2-deoxy-2-phtha-
limido- -glucopyranose (23).—Ceric ammo-
nium nitrate (CAN, 378 mg, 0.69 mmol) was
D
-manno-
2-O-Acetyl-3,4,6-tri-O-benzyl-h-
pyranosyl - (1“3) - [3,4,6 - tri - O - benzyl - 2 - de-
oxy-2-phthalimido-i- -glucopyranosyl-(1“2)]-
4,6-O-di-tert-butylsilanediyl-i- -mannopyra-
nosyl - (1“4) - 3,6 - di - O - benzyl - 2 - deoxy - 2-
phthalimido - i - - glucopyranosyl - (1“4)-
D
-manno-
D
D
D
D
D
D

274
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
cessively with water and brine. The organic
[2,3,4-tri-O-benzyl-h- -fucopyranosyl-(1“6)]-
L
layer was dried over Na2SO4 and evaporated
in vacuo. The residue was successively purified
by silica gel column chromatography (1:1
EtOAc–toluene) and preparative TLC (1:1
EtOAc–toluene) to afford compound 27 (39.7
3 - O - benzyl - 2 - deoxy - 2 - phthalimido - i -
D
-
glucopyranosyl azide (26).—To a precooled
(−20 °C) mixture of compounds 24 (62.9 mg,
0.03 mmol) and 25 (123.4 mg, 0.15 mmol) in
,
dry CH2Cl2 (1.5 mL) containing 4A MS (100
1
mg, 90%); [h]D +41.1° (c 0.75, CHCl3); H
mg), BF3·OEt2 (5 mL, 0.04 mmol) was added
and the mixture was stirred at the same tem-
perature. After 2 h an additional amount of
BF3·OEt2 (2.5 mL, 0.02 mmol) was added, and
the mixture was stirred for one additional
hour. The mixture was quenched with aq
NaHCO3, diluted with EtOAc, and filtered
through Celite. The filtrate was washed suc-
cessively with water, aq NaHCO3 and brine,
dried over Na2SO4, and evaporated in vacuo.
The residue was subjected to a column of
Bio-beads SX-1 (toluene), and hexasaccharide-
containing fractions were further purified by
silica gel column chromatography (5–10%
EtOAc in toluene) to afford compound 26
(31.8 mg, 38%); [h]D +23.4° (c 0.32, CHCl3);
1H NMR (270 MHz, C6D6): l 7.82–6.61 (m,
Ar), 6.01 (brs, 1 H, H-2IV), 5.85 (d, 1 H, J 8.2
Hz, H-1), 5.81 (d, 1 H, J 7.3 Hz, H-1), 5.61 (s,
1 H, H-1IV), 5.34–5.26 (m, 5 H), 5.14 (d, 1 H,
J 10.9 Hz, benzylic), 1.70 (s, 3 H, OAc), 1.29
(s, 9 H, t-Bu), 1.25 (d, 3 H, J 6.4 Hz, H-6VIII),
1.03 (s, 9 H, t-Bu); 13C NMR (67.5 MHz,
C6D6): l 105.1, 100.5, 97.9, 97.4, 85.5, 83.2,
80.7, 80.1, 79.3, 78.8, 78.6, 78.4, 77.2, 76.6,
75.8, 75.2, 74.9, 74.8, 74.5, 74.4, 73.9, 73.7,
73.6, 73.3, 72.9, 72.7, 71.9, 71.7, 69.9, 69.3,
67.0, 63.9, 57.7, 57.5, 56.2, 28.2, 27.5, 23.0,
20.7, 20.2, 17.0; HRFABMS: Calcd for
C154H160N6NaO33Si [M+Na]+: 2672.0693:
Found 2672.0706.
NMR (270 MHz, C6D6): l 7.93–6.62 (m, Ar),
6.24 (brs, 1 H, H-2IV), 6.10 (brs, 1 H, H-1IV),
5.97 (d, 1 H, J 8.4 Hz, H-1), 5.93 (d, 1 H, J
9.2 Hz, H-1), 5.45 (d, 1 H, J 12.9 Hz, ben-
zylic), 5.37 (d, 1 H, J 13.0 Hz, benzylic), 5.29
(d, 1 H, J 9.2 Hz, H-1), 5.24–5.19 (m, 2 H,
H-1, benzylic), 3.05 (m, 1 H), 2.78 (d, 1 H, J
10.0 Hz, OH), 2.16 (s, 3 H, OAc), 1.23 (d, 3
H, J 6.4 Hz, H-6VIII); 13C NMR (67.5 MHz,
C6D6): l 104.6, 97.76 (×2), 97.66, 97.2, 96.4;
HRFABMS: Calcd for C146H144N6NaO33
[M+Na]+: 2531.9672. Found: 2531.9763.
2-O-Acetyl-3,4,6-tri-O-benzyl-h-
pyranosyl - (1“6) - [2 - O - acetyl - 3,4,6 - tri - O-
benzyl-h- -mannopyranosyl-(1“3)]-[3,4,6-tri-
O-benzyl-2-deoxy-2-phthalimido-i- -glucopy-
ranosyl-(1“2)]-i- -mannopyranosyl-(1“4)-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-i-
glucopyranosyl-(1“4)-[2,3,4-tri-O-benzyl-h-
-fucopyranosyl-(1“6)]-3-O-benzyl-2-deoxy-
2-phthalimido-i- -glucopyranosyl azide (28).
D
-manno-
D
D
D
D-
L
D
—Compounds 27 (18.0 mg, 7.2 mmol) and 14
,
(4.6 mg, 9.0 mmol) and 4A MS (1.0 g) were
suspended in dry CH2Cl2 (2.0 mL). Silver
triflate (3.0 mg, 11.0 mmol) was added, and
the mixture was stirred for 1.5 h. The reaction
was quenched with aq Na2CO3, diluted with
EtOAc, and filtered through Celite. The
filtrate was dried over MgSO4 and the solvent
was evaporated in vacuo. The residue was
subjected to a column of Bio-beads SX-3 (3:1
toluene–CHCl3) and heptasaccharide-contain-
ing fractions were further purified by prepara-
tive TLC (1:3 EtOAc–toluene) to afford
compound 28 (7.2 mg, 34%), the correspond-
ing regioisomer (4.3 mg, 20%) and recovered
27 (2.8 mg, 16%).
2-O-Acetyl-3,4,6-tri-O-benzyl-h-
pyranosyl - (1“3) - [3,4,6 - tri - O - benzyl - 2 - de-
oxy-2-phthalimido-i- -glucopyranosyl-(1“2)]-
i- -mannopyranosyl-(1“4)-3,6-di -O-benzyl-
2-deoxy-2-phthalimido-i- -glucopyranosyl-
(1“4) - [2,3,4-tri-O-benzyl-h- -fucopyranosyl-
(1“6)]-3-O-benzyl-2-deoxy-2-phthalimido-i-
-glucopyranosyl azide (27).—Tributylammo-
D-manno-
D
D
D
L
D
Compound 28: [h]D +39.7° (c 0.36,
1
nium hydrogen fluoride (1.6 M solution in
THF, 0.1 mL, 0.16 mmol) was added to a
solution of compound 26 (46.4 mg, 17.5
mmol), and the solution was stirred at rt for 1
h. The resulting mixture was quenched with
water, diluted with EtOAc, and washed suc-
CHCl3); H NMR (500 MHz, C6D6): l 7.98–
6.63 (m, 87 H, Ar), 6.23 (dd, 1 H, J 2.6 and
1.5 Hz, H-2IV or H-2V), 6.09 (brs, 1 H, H-1),
5.95 (d, 1 H, J 8.3 Hz, H-1), 5.90 (d, 1 H, J
8.3 Hz, H-1), 5.69 (d, 1 H, J 2.8, 1.5 Hz, H-2V
or H-2IV), 5.43 (d, 1 H, J 12.9 Hz, benzylic),

Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
275
24.1, 23.4, 23.1, 17.1; HRFABMS: Calcd for
5.37 (d, 1 H, J 12.7 Hz, benzylic), 5.31 (d, 1
H, J 9.3 Hz, H-1), 5.26–5.22 (m, 2 H, H-1,
benzylic), 5.07–4.86 (m, 7 H), 4.79–3.73 (m,
54), 3.63 (dd, 1 H, J 10.0 and 7.7 Hz), 3.57–
3.46 (m, 3 H), 3.39 (brs, 1 H), 3.19–3.15 (m, 2
H), 2.86 (d, 1 H, J 9.8 Hz, OH), 2.00 (s, 3 H,
OAc), 1.72 (s, 3 H, OAc), 1.25 (d, 3 H, J 6.3
Hz, H-6VIII); 13C NMR (125 MHz, C6D6): l
105.3, 97.81, 97.75, 97.4, 85.5, 84.0, 80.4,
80.24, 80.18, 79.2, 79.1, 78.5, 77.1, 76.6, 76.1,
75.9, 75.3, 75.1, 74.9, 73.6, 73.4, 73.3, 73.23,
73.17, 72.9, 72.4, 72.1, 71.8, 70.1, 69.9, 69.5,
69.4, 69.0, 66.9, 57.5, 56.9, 56.1, 30.2, 20.9,
C153H171N6NaO34
Found: 2636.1846.
[M+Na]+:
2636.1836.
N2-Benzyloxycarbonyl-N4-{3,4,6-tri-O-ben-
zyl-h-
benzyl-h-
amido - 3,4,6 - tri - O - benzyl - 2 - deoxy - i -
glucopyranosyl-(1“2)]-i- -mannopyranosyl-
(1“4)-acetamido-3,6-di-O-benzyl-2-deoxy-
i- -glucopyranosyl-(1“4)-O- [2,3,4-tri-O-
benzyl-h- -fucopyranosyl-(1“6)]-2-acetamido-
3-O-benzyl-2-deoxy-i- -glucopyranosyl}-
D
-mannopyranosyl-(1“6)-[3,4,6-tri-O-
D
-mannopyranosyl-(1“3)]-[2-acet-
D
-
D
D
L
D
L-
asparagine benzyl ester (32).—DCC (8.8 mg,
43 mmol) was added to a solution of Z-Asp-
OBn (25.8 mg, 72.2 mmol) in dry CH2Cl2 (1.5
mL), and the mixture was stirred at 0 °C for
30 min. The precipitate was filtered off, and
the filtrate was dissolved in EtOAc (1.0 mL).
A solution of compound 30 (5.0 mg, 1.9 mmol)
in MeOH (1.0 mL) was added, followed by
Lindlar catalyst. The mixture was stirred un-
der a H2 atmosphere at rt for 5 h and then
filtered through a membrane filter. The filtrate
was evaporated in vacuo and purified by a
column of Bio-beads SX-3 (3:1 toluene–
CHCl3). The asparagine-linked heptasaccha-
ride-containing fractions were further purified
by preparative TLC (1:9 MeOH–CHCl3) to
20.7,
16.8;
HRFABMS:
Calcd
for
C146H144N6NaO33
[M+Na]+:
2531.9672.
Found: 2531.9763.
3,4,6 - Tri - O-benzyl- h -
(1“6)-[3,4,6-tri-O-benzyl-h- -mannopyra-
D
- mannopyranosyl-
D
nosyl - (1“3)] - [2 - acetamido - 3,4,6 - tri-O-
benzyl-2-deoxy - i - - glucopyranosyl-(1“2)]-
i- -mannopyranosyl-(1“4) - O - 2 - acetamido-
3,6-di-O-benzyl-2-deoxy-i- -glucopyranosyl-
(1“4)-[2,3,4-tri-O-benzyl-h- -fucopyranosyl-
(1“6)]-2-acetamido-3-O-benzyl-2-deoxy-i-
-glucopyranosyl azide (30).—Compound 28
D
D
D
L
D
(9.8 mg, 3.3 mmol) was treated with ethylene-
diamine (1.0 mL) in EtOH (3 mL) under
reflux. After 16 h an additional amount of
ethylenediamine (0.2 mL) was added and the
mixture was stirred at the same temperature
for 5 h. The resulting mixture was evaporated
in vacuo, coevaporated with toluene (×3),
and was dissolved in MeOH (3 mL). The
solution was treated at 0 °C with Ac2O (0.75
mL) for 1.5 h and evaporated in vacuo. The
residue was subjected to a column of Bio-
beads SX-3 (3:1 toluene–CHCl3) to afford
compound 30 (8.1 mg, 94%); [h]D −12.6° (c
1
afford compound 32 (3.5 mg, 63%); H NMR
(600 MHz, C6D6): l 7.52–6.98 (m, Ar), 6.33
(brs, 1 H, NH), 5.81 (s, 1 H, H-1IV), 5.72 (d, 1
H, J 7.8 Hz, H-1), 5.23 (brs, 1 H, H-1), 5.15
(d, 1 H, J 11.2 Hz, benzylic CH2), 3.15 (brs, 1
H), 3.04 (brs, 1 H, H-2), 2.75 (brs, 1 H,
CHA2 sn), 2.53 (brs, 1 H, CHA2 sn), 2.09 (s, 3 H,
NAc), 1.75 (s, 3 H, NAc), 1.71 (s, 3 H, NAc),
1.33 (d, 3 H, J 5.9 Hz, H-6VIII).
N4 - {h -
D
- mannopyranosyl - (1“6) - [h -
mannopyranosyl - (1“3)] - [2 - acetamido - 2 - de-
oxy-i- -glucopyranosyl-(1“2)]-i- -manno-
pyranosyl-(1“4)-2-acetamido-2-deoxy-i-
glucopyranosyl - (1“4) - [h - - fucopyranosyl-
(1“6)]-2-acetamido-2-deoxy-i- -glucopyra-
nosyl}- -asparagine (1a).—Compound 32 (3.2
D
-
1
0.54, CHCl3); H NMR (400 MHz, C6D6): l
D
D
7.57–6.60 (m, Ar), 6.53 (brs, 1 H, NH), 6.34
(s, 1 H, H-1), 5.98 (brs, 1 H, NH), 5.83 (s, 1
H, H-1), 5.69 (d, 1 H, J 8.3 Hz, H-1), 3.16 (d,
1 H, J 9.0 Hz), 3.00 (m, 1 H, H-2), 2.07 (s, 3
H, NAc), 1.76 (s, 3 H, NAc), 1.70 (s, 3 H,
NAc), 1.30 (d, 3 H, J 6.3 Hz, H-6VIII); 13C
NMR (100 MHz, C6D6): l 104.0, 99.8, 98.7,
98.1, 97.9, 88.75, 81.7, 81.0, 80.6, 79.7, 79.32,
79.26, 79.1, 79.0, 78.2, 77.2, 76.9, 76.2, 75.5,
74.9, 74.7, 73.8, 73.6, 73.4, 73.1, 73.0, 72.9,
72.5, 72.3, 72.0, 71.8, 71.1, 70.8, 70.3, 69.8,
69.6, 67.5, 67.3, 63.8, 63.5, 60.3, 55.5, 30.3,
D-
L
D
L
mg, 1.1 mmol) was hydrogenated over
Pd(OH)2–C (3.5 mg) in 4:2:1 EtOH–THF–
water (1.4 mL) at rt for 62 h. The resulting
mixture was filtered through a membrane
filter, and the filtrate was evaporated in vacuo.
The residue was passed through a C18 re-
versed-phase cartridge (water) to afford, after
lyophilization, compound 1a (1.0 mg, 61%);

276
Y. Ohnishi et al. / Carbohydrate Research 328 (2000) 263–276
1H NMR (500 MHz, D2O, 50 °C): l 5.12 (d, 1
H, J 1.2 Hz, H-1IV), 5.06 (d, 1 H, J 9.7 Hz,
H-1I), 4.89 (1 H, J 2.2 Hz, H-1IV), 4.87 (1 H,
J 3.7 Hz, H-1F), 4.76 (brs, 1 H, H-1II), 4.73 (d,
1 H, J 8.9 Hz, H-1II or H-1IX), 4.71 (d, 1 H, J
7.5 Hz, H-1IX or H-1II), 2.71 (dd, 1 H, J 16
and 4.9 Hz, CHA2 sn), 2.45 (dd, 1 H, J 16 and
8.9 Hz, CHA2 sn), 2.08 (s, 3 H, NAc), 2.03 (s, 3
H, NAc), 2.01 (s, 3 H, NAc), 1.20 (d, 3 H, J
6.8 Hz, H-6VIII).
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We are grateful to Dr Jun Uzawa and his
staff for NMR measurements. We thank Dr
Tomoya Ogawa for his encouragement
throughout this work and Ms A. Takahashi
for technical assistance.
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