Synthetic studies on glycosphingolipids from the parasite Echinococcus multilocularis

Article abstract of DOI:10.1016/S0008-6215(99)00027-0

Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be expected to be involved in host-parasite interactions. We have synthesized these glycosphingolipid analogues containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21, and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol and subsequently deacylated give four target glycosphingolipid analogues. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00027-0

Carbohydrate Research 316 (1999) 58–70
Synthetic studies on glycosphingolipids from the parasite
Echinococcus multilocularis
Noriyasu Hada, Eriko Hayashi, Tadahiro Takeda *
Kyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, 105-8512 Tokyo, Japan
Received 23 November 1998; accepted 19 January 1999
Abstract
Novel neutral glycosphingolipids isolated from the metacestodes of Echinococcus multilocularis by Persat, may be
expected to be involved in host–parasite interactions. We have synthesized these glycosphingolipid analogues
containing 2-branched fatty alkyl residues in place of ceramide. The glycosylation of galactosyl donors 4 and 5 with
each of the acceptors 2 and 11 in the presence of N-iodosuccinimide (NIS)/TfOH, and the glycosylation of fucosyl
donor 13 with acceptors 12 and 20 in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) gave the
desired oligosaccharide derivatives at good yield. The fully per-O-acylated 2-(trimethylsilyl)ethyl glycosides 6, 15, 21,
and 26 were converted to glycosylimidates 7, 16, 22, and 27, which were condensed with 2-(tetradecyl)hexadecanol
and subsequently deacylated give four target glycosphingolipid analogues. © 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Glycosphingolipids; Echinococcus mululocularis; Chemical synthesis
We have been interested in the structure
1. Introduction
and biological function of glycolipids from
invertebrate animal species and have so far
synthesized an octasaccharide of the Hyriopsis
schlegelii glycosphingolipid in order to eluci-
date the mechanism of its fertilization [8]. We
have now turned our attention to a glycolipid
from a parasite that may be involved in host–
parasite interactions such as species-related
infestation and stage development or the
choice of target organs that parasites preferen-
tially invade. There are some reports that
patients infected with Taenia [9], Leishmania
[10], or Schistosoma [11] were shown to ex-
press antibodies directed against parasite gly-
colipid. However, the structural characteristics
of these immunogenic glycolipids have not
been established.
Over the past few years we have seen great
interest in the preparation of oligosaccharides
containing sialic acids, such as sialyl Lewis X
in mammalian membranes, in order to eluci-
date the biological function of carbohydrates
on the cell surface [1,2]. A number of sialyl-
oligosaccharides and their mimetics have been
synthesized by many carbohydrate chemists
[3–5]. On the other hand, synthetic studies of
oligosaccharides from invertebrate animal spe-
cies that do not have gangliosides have been
neglected, in spite of their having interesting
structures [6,7].
* Corresponding author. Fax: +81-3-5400-2556.
E-mail address: takeda-td@kyoritsu-ph.ac.jp (T. Takeda)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00027-0

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
59
Table 1
2. Results and discussion
Major glycosphingolipid structures in E. multilocularis
metacestodes
Syntheses of monosaccharide deri6ati6es.—
Syntheses of the additional galactopyranosyl
building blocks 2 and 4 were carried out as
depicted in Scheme 1. 2-(Trimethylsilyl)ethyl
b-
a-
b-
b-
D
-Galp-(1“6)-b-
D
-Galp-(1l1)-Cer
-Galp-(1“6)-b- -Galp-(lll)-Cer
-Galp-(l“6)-b- -Galp-(lll)-Cer
-Fucp-(1“3)]-b- -Galp-(1“6)-b-
A
B
C
D
L
-Fucp-(1“3)-b-
D
D
D
D
D
-Galp-(1“6)-b-
-Galp-(1“6)-[a-
-Galp-(lll)-Cer
D
D
L
D
2,3,4-tri-O-benzoyl-b-
was prepared from known 2-(trimethylsil-
yl)ethyl b- -galactopyranoside (1) [14] follow-
D
-galactopyranoside (2)
D
ing a three-step procedure. Regioselective silyl
ation of the starting material with tert-
butyldimethylsilyl chloride (TBDMS-Cl), fol-
lowed by benzoylation and subsequent acid
hydrolysis of the silyl group, gave compound
2. On the other hand, donor 4 was obtained
Recently, Persat et al. have reported gly-
cosphingolipids with b- -Galp-(1“6)- -Galp
D
D
sequences in metacestodes of the parasite,
Echinococcus mululocularis (Table 1) [12]. In
this study they demonstrated for the first time
that metacestodes of E. multilocularis con-
tained glycolipids that were recognized by
serum antibodies from different patients with
alveolar hydatid disease [12].
from phenyl 1-thio-b- -galactopyranoside (3)
D
[15] by regioselective benzylation of the in situ
prepared stannylidene derivative [16] of 3 with
benzyl bromide and subsequent regioselective
silylation with TBDMS-Cl, followed by
acetylation.
The glycolipids related to this binding were
specifically bound to the neutral glycosphin-
golipid. It is interesting that no reaction was
observed with acid glycolipids such as GM1,
GM3 and GD1a. In addition, fucose has been
shown to form at least seven types of linkages,
but none is of the two novel fucolipid struc-
tures of the a-(1“3)-Galp type. The two
Systematic syntheses of the each of the gly-
colipid analogues.—In this work, four kinds of
target glycolipid analogues 9, 18, 24 and 29
were synthesized by stepwise condensation
[17] of suitably protected monosaccharide
units. A systematic plan for these compounds
was designed as shown in Schemes 2 and 3.
First, we chose compounds 6, 15, 21 and 26,
which were the per-O-acylated 2-(trimethylsil-
yl)ethyl glycoside derivatives, as the intermedi-
ates. The glycosylation of 2 with phenyl
novel fucolipids have structures a-
(1“3)-b- -Galp-(1“6)-b- -Galp-(1l1)-Cer
and b- -Galp-(1“6)-[a- -Fucp-(1“3)]-b-
Galp-(1“6)-b- -Galp-(1l1)-Cer. The oli-
L
-Fucp-
D
D
D
L
D
-
D
gosaccharides of four glycolipids were the
target for the synthetic studies described
herein as part of our investigation into syn-
thetic oligosaccharides of structural and bio-
logical interest. We chose a systematic,
integrated approach for the synthesis of te-
trasaccharides and have synthesized these gly-
colipid analogues containing 2-branched fatty
alkyl residues in place of ceramide [13].
2,3,4,6-tetra-O-benzoyl-1-thio-b- -galactopy-
D
ranoside (5) [18] in the presence of N-iodosuc-
,
cinimide (NIS)/TfOH (cat.) [19] and 4 A
molecular sieves in dichloromethane for 30
min at 0 °C gave the desired disaccharide 6
1
(70%) as evidenced by H NMR spectroscopy
(H-1% 4.91 ppm, J 7.9 Hz). On the other hand,
the common donor 4 was chosen for the
Scheme 1.

60
N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
Scheme 2.
target compounds 15, 21 and 26, which was
condensed with 2 under the agency of NIS/
TfOH (cat) to yield disaccharide 10 (58%) as
pound 11, a part of which was converted by
O-acetylation with Ac2O and followed by hy-
drogenation over 10% Pd–C to give the disac-
charide derivative 12. The glycosylation of
compound 12 with methyl 2,3,4-tri-O-benzyl-
1
evidenced by H NMR spectroscopy (H-1%
4.42 ppm, J 8.0 Hz). Selective removal of the
TBDMS group in 10 with 3:1:1 AcOH–
THF–H2O gave the partially protected com-
1-thio-b-
L
-fucopyranoside (13) [3] in the pres-
ence of dimethyl(methythio)sulfonium triflate
Scheme 3.

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
61
Table 2
13C NMR data (l) for selected compounds
Carbon atom
Compound
6
10
14
19
25
C-l(GalI)
2
3
4
101.1
69.6
71.9
68.6
73.0
67.6
100.9
69.8
71.7
68.0
71.3
61.7
101.0
69.9
72.0
68.7
73.1
67.8
101.2
70.6
77.0
65.5
73.7
60.6
100.9 (157.3)
69.8
71.9
68.6
73.1
67.4
101.0 (161.4)
70.7
75.4
69.0
101.0
69.9
72.0
68.6
72.7
67.4
101.0
70.2
76.8
65.8
72.2
66.8
101.1 (159.3)
70.0
72.0
68.5
73.2
67.5
101.1 (161.4)
70.9
77.3
69.4
5
6
C-1 (GalII)
2
3
4
5
71.4
61.9
72.6
67.1
6
C-1 (Fuc)
99.4 (169.7)
75.8
78.6
74.7
77.4
99.1 (171.7)
750.8
78.7
74.9
77.7
16.5
101.2 (159.3)
69.8
71.6
68.1
2
3
4
5
6
16.4
C-1 (GalIII
)
101.0
69.8
71.7
68.0
71.4
62.0
2
3
4
5
6
71.3
62.0
Si(tert-Bu)(CH3)2
CH2CH2Si(CH3)3
CH2Ph
137.7
25.7
−5.6
67.7
18.0
−1.4
71.2
67.5
17.5
−1.5
67.5
18.0
−1.5
74.7
73.0
72.8
67.7
18.0
−1.5
71.2
67.7
18.0
−1.5
74.7
72.9
72.8
1
derivative 19 (78%), as evidenced by H NMR
spectroscopy (H-1¦ 4.84 ppm, J 7.9 Hz). Re-
moval of the 3%-O-benzyl group from 19 by
catalytic hydrogenolysis over Pd–C gave the
accepter 20 (82%) for the tetrasaccharide
derivative, and subsequent acetylation gave
the trisaccharide 21. Furthermore the glycosy-
lation of 20 with 13 in the presence of
DMTST as described for 14 gave the tetrasac-
charide derivative 25 (90%), as evidenced by
1H NMR spectroscopy (H-1 of Fuc 4.96 ppm,
J 3.7 Hz). Then 25 was converted by O-debenz-
ylation and subsequent acetylation, to give the
tetrasaccharide 26.
(DMTST) [20] as the glycosyl promoter and 4
A molecular sieves in dichloromethane for 5 h
at 0 °C gave the desired a-glycoside 14 in 89%
yield. Significant signals of the fucose unit in
the 1H NMR spectrum showed a signal for the
anomeric hydrogen atoms at l 4.96 (d, J 3.7
,
Hz). The a-
L
configuration of the newly
formed glycosidic bond was also indicated by
the JC,H value of 169.7 Hz in the 13C NMR
spectrum. Catalytic hydrogenolysis (10%, Pd–
C) in methanol–AcOH of the benzyl groups
in 14 and subsequent O-acetylation gave the
trisaccharide 15 (81%) (Table 2).
On the other hand, the glycosylation of 11
with 5 in the presence of NIS/TfOH as de-
scribed for compound 6 gave the trisaccharide
Next, for the selective removal of the 2-
(trimethylsilyl)ethyl group, the fully acylated

62
N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
oligosaccharides 6, 15, 21 and 26 were treated
[21] with trifluoroacetic acid in dichloro-
methane for 30 min at 0 °C to give the 1-hy-
droxy compounds, which, on further
treatment with trichloroacetonitrile in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in dichloromethane for 2 h at 0 °C,
gave the corresponding receptor carbohy-
drates 7, 16, 22 and 27. Glycosylation of
2-(tetradecyl)hexadecanol with each of the
glycosyl donors, which was carried out in the
presence of BF3·OEt and AW300 molecular
sieves for 5 h at 0 °C, afforded the desired
b-glycosides 8 (36%), 17 (37%), 23 (49%) and
28 (29%) in low yield. Following this lead, we
attempted a similar coupling of donor 27 with
the acceptor, 2-(tetradecyl)hexadecanol, in the
presence of Me3SiOTf to give 28 in a more
acceptable yield of 53%. Finally, removal of
all acyl groups with sodium methoxide in 3:1
methanol–1,4-dioxane for 5 h at room tem-
perature afforded the desired glycolipid (A–
D) analogues (9, 18, 24 and 29). Biological
results using sera from patients with alveolar
hydatid disease for these compounds will be
reported in detail elsewhere.
(1) (5.0 g, 17.8 mmol) in DMF (30 mL) were
added at 0 °C imidazole (2.42 g, 35.6 mmol)
and tert-butyldimethylsilyl chloride (2.82 g,
18.7 mmol). The reaction mixture was stirred
at room temperature (rt) for 6 h, then diluted
with CHCl3 and washed with 5% HCl, aq
NaHCO3 and water, dried (Na2SO4), and con-
centrated. The product was purified by silica
gel column chromatography using 10:1
CHCl3–MeOH as eluent to give 2-(tri-
methylsilyl)ethyl-6-O-(tert-butyldimethylsilyl)-
b- -galactopyranoside (5, 87 g, 83.1%). To a
D
solution of this compound in pyridine (50 mL)
was added benzoyl chloride (7 mL), and the
mixture was stirred for 4 h at 0 °C. Extractive
isolation gave 2-(trimethylsilyl)ethyl 6-O-(tert-
butyldimethylsilyl)-2,3,4-tri-O-benzoyl-b- -
D
galactopyranoside quantitatively. This com-
pound was treated with 80% AcOH (50 mL)
at 40 °C for 3 h, and following removal of
acetic acid and water by coevaporation with
toluene, was purified by column chromatogra-
phy (3:2 hexane–EtOAc) on silica gel to give
compound 2 (9.37 g, 89%): [h]D+180° (c 0.9
1
CHCl3); H NMR (CDCl3): l 8.12–7.22 (m,
15 H, 3×Ph), 5.84 (dd, 1 H, J1,2 7.9 Hz, J2,3
10.3 Hz, H-2), 5.82 (d, 1 H, J3,4 2.4 Hz, H-4),
5.58 (dd, 1 H, H-3), 4.83 (d, 1 H, H-1),
4.12–4.02 (m, 2 H, H-6a, –OCH2CH2–), 3.85
(ddd, 1 H, H-5), 3.67–3.62 (m, 2 H, H-6b,
–OCH2CH2–), 2.71 (t, 1 H, OH), 0 97–0 86
(m, 2 H, –OCH2CH2–), −0.06 (s, 9 H,
Si(CH3)3); Anal. Calcd for C32H36O9Si (592.7)
C, 64.85; H, 6.12. Found: C, 64.59; H, 6.10.
Phenyl 2,4-di-O-acetyl-3-O-benzyl-6-O-(tert-
3. Experimental
General.—Optical rotations were deter-
1
mined with a Jasco digital polarimeter. H
NMR and 13C NMR spectra were recorded
with a JNM A 500 FT NMR spectrometer
with Me4Si as the internal standard for solu-
tions in CDCl3. MALDI-TOFMS was
recorded on a Perceptive Voyager RP mass
spectrometer. TLC was performed on Silica
Gel 60 F254 (E. Merck) with detection by
quenching of UV fluorescence and by spraying
with 10% H2SO4. Column chromatography
was carried out on Silica Gel 60 (E. Merck).
butyldimethylsilyl)-1-thio-i-
oside (4).—A mixture of phenyl 1-thio-b-
D
-galactopyran-
D-
galactopyranoside (3) (500 mg, 2.08 mmol),
dibutyltin oxide (570 mg, 2.30 mmol) and 30
mL of dry benzene was stirred under reflux for
5 h. Benzene (15 mL) was distilled off, and the
solution was cooled to 50 °C and treated with
Bu4NBr (804 mg) and BnBr (0.6 mL). After
the reaction mixture was stirred for 5 h, the
solution was concentrated. Purification of the
residue by column chromatography (10:1
CHCl3–MeOH) on silica gel gave phenyl 3-O-
2-(Trimethylsilyl)ethyl b-
(1), phenyl 1-thio-b- -galactopyranoside (3),
phenyl 2,3,4,6-tetra-O-benzoyl-1-thio-b-
galactopyranoside (5) and methyl 2,3,4-tri-O-
D
-galactopyranoside
D
D
-
benzyl-1-thio-b- -fucopyranoside (13) were
L
prepared by literature methods [14,15,18,3].
benzyl-1-thio-b- -galactopyranoside (374 mg,
D
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-
50%). To a solution of this compound (583
mg, 1.61 mmol) in DMF (7 mL) were added
i-
D
-galactopyranoside (2).—To a solution of
2-(trimethylsilyl)ethyl b-
D
-galactopyranoside

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
63
EtOAc as eluent to give 6 (416 mg, 70%):
[h]D +63.2° (c 0.2, CHCl3); 1H NMR
(CDCl3): l 8.11–7.21 (m, 35 H, 7×Ph), 5.94
(d, 1 H, J3,4 3.7 Hz, H-4%), 5.92 (d, 1 H, J3%,4%
3.7 Hz, H-4%), 5.77 (dd, 1 H, J1%,2%, 7.9 Hz, J2%,3%
10.4 Hz, H-2%), 5.72 (dd, 1 H, J1,2 7.9 Hz, J2,3
10.4 Hz, H-2), 5.57 (dd, 1 H, H-3%), 5.51 (dd,
1 H, H-3), 4.91 (d, 1 H, H-1%), 4.70 (d, 1 H,
H-1), 4.39 (dd, 1 H, J5%,6%a, 8.5 Hz, J6%a,6%b 13.5
Hz, H-6a%), 4.27–4.23 (m, 2 H, H-5%,6b%),
4.20–4.16 (m, 2H, H-5, 6a), 3.94–3.89 (m, 2
H, –CH2CH2Si, H-6b), 3.46 (dt, 1 H, –
CH2CH2Si), 0.85–0.71 (m, 2 H, –CH2CH2Si),
−0.08 (s, 9 H, Si(CH3)3); Anal. Calcd for
C66H62O18Si (1171.3): C, 67.68; H, 5.34.
Found: C, 67.63; H, 5.31.
imidazole (219 mg, 3.22 mmol) and tert-
butyldimethylsilyl chloride (292 mg, 1.93
mmol) at 0 °C. The reaction mixture was
stirred at rt for 3 h and then diluted with
CHCl3, washed with 5% HCl, aq NaHCO3
and water, dried (Na2SO4), and concentrated.
The product was chromatographed on silica
gel using 2:1 hexane–EtOAc as eluent to
provide phenyl 3-O-benzyl-6-O-(tert-butyl-
dimethylsilyl)-1-thio-b- -galactopyranoside.
D
To a solution of this compound in pyridine
(20 mL) was added acetic anhydride (15 mL),
and the mixture was stirred for 4 h at 0 °C.
The reaction mixture was poured into ice-wa-
ter and extracted with CHCl3. The extract was
washed sequentially with 5% HCl, aq
NaHCO3 and water, dried (Na2SO4), and con-
centrated. The product was purified by silica
gel column chromatography using 4:1 hex-
ane–ethyl acetate as eluent to give 4 (830 mg,
2,3,4,6-Tetra-O-benzoyl-i-
D
-galactopyran-
-galacto-
osyl-(1“6)-2,3,4-tri-O-benzoyl-h-
D
pyranosyl trichloroacetimidate (7).—To a solu-
tion of 6 (52 mg, 44 mmol) in CH2Cl2 (2.5
mL) cooled to 0 °C was added CF3COOH (1.5
mL), and the mixture was stirred for 30 min.
at rt and concentrated. Ethyl acetate and
toluene (1:2) were added and then evaporated
to give the 1-hydroxy compound. To a solu-
tion of the residue in CH2Cl2 (0.8 mL) cooled
to 0 °C were added trichloroacetonitrile (132
mL 132 mmol) and DBU (7.3 mL 48 mmol).
The mixture was stirred for 2 h at 0 °C. After
completion of the reaction, the mixture was
concentrated. Column chromatography (3:2
hexane–EtOAc) of the residue on silica gel
gave 7 (38 mg, 70%) as an amorphous mass:
[h]D +116.9° (c 1.0, CHCl3); 1H NMR
(CDCl3): l 8.33 (s, 1 H, NH), 8.09–7.20 (m,
35 H, 7×Ph), 6.76 (d, 1 H, J1,2 3.7 Hz, H-1),
6.08 (d, 1 H, J3,4 3.1 Hz, H-4), 6.01 (dd, 1 H,
J2,3 10.0 Hz, H-3), 5.92 (d, 1 H, J3%,4% 3.1 Hz,
H-4%), 5.85 (dd, 1 H, J2,3 10.4 Hz, H-2), 5.74
(dd, 1 H, J1%,2% 7.9 Hz, J2%,3% 10.4 Hz, H-2%), 5.53
(dd, 1 H, H-3%), 4.90 (d, 1 H, H-1%), 4.69–3.88
(m, 6 H, H-5, 5%, 6a, 6b, 6%a, 6%b); MALDI-
TOFMS: Calcd for C63H50Cl3NO18: m/z
1214.7. Found: m/z 1237.5 [M+Na]+, 1253.7
[M+K]+.
1
92%, 2 steps): [h]D +55.0° (c 0.6, CHCl3); H
NMR (CDCl3): l 7.49–7.25 (m, 10 H, 2×
Ph), 5.61 (d, 1 H, J3,4 3.1 Hz, H-4), 5.16 (t, 1
H, J1,2,2,3 9.7 Hz, H-2), 4.71 and 4.42 (each d,
2 H, Jgem 12.2 Hz, benzylic methylene), 4.64
(d, 1 H, H-1), 3.74 (dd, 1 H, J5,6a 9.2 Hz, J6a,6b
11.2 Hz, H-6a), 3.67–3.64 (m, 2 H, H-5, H-
6b), 3.55 (dd, 1 H, H-3), 2.12 and 2.05 (each s,
6 H, 2×OAc), 0.88 (s, 9H, 3×CH3), −0.04
(s, 6 H, –Si(CH3)2–); Anal. Calcd for
C29H40O7SSi (560.8): C, 62.11; H, 7.19.
Found: C, 61.85; H, 7.25.
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
oyl-i-
benzoyl-i-
solution of 2 (300 mg, 0.51 mmol) and phenyl
-galactopyr-
D
-galactopyranosyl(1“6)-2,3,4-tri-O-
D
-galactopyranoside (6).—To
a
2,3,4,6-tetra-O-benzoyl-1-thio-b-
D
anoside 5 (421 mg, 0.61 mmol) in dry CH2Cl2
,
(5 mL) was added powdered 4 A molecular
sieves (1 g), and the mixture was stirred for 2
h at rt, then cooled to 0 °C. N-Iodosuccin-
imide (NIS; 160 mg, 0.73 mmol) and trifl-
uoromethanesulfonic acid (TfOH; 5.3 mL 0.06
mmol) were added to the mixture, which was
stirred for 30 min at 0 °C, then neutralized
with Et3N. The solids were filtered off and
washed with CHCl3. The combined filtrate
and washings were successively washed with
aq Na2S2O3 and water, dried (Na2SO4), and
concentrated. The product was chro-
matographed on silica gel using 3:2 hexane–
2-(Tetradecyl)hexadecyl
benzoyl-i- -galactopyranosyl- (1“6)-2,3,4-
tri-O-benzoyl-i- -galactopyranoside (8).—To
2,3,4,6-tetra-O-
D
D
a solution of the trichloroacetimidate 7 (90
mg, 74 mmol) and 2-(tetradecyl)hexadecanol
(65 mg, 148 mmol) in CH2Cl2 (1 mL) were

64
N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
(630 mg, 2.82 mmol) and TfOH (50 mL, 0.56
mmol) were added to the mixture, which was
stirred for 20 min at 0 °C, then neutralized
with Et3N. The solids were filtered off and
washed with CHCl3. The combined filtrate
and washings was successively washed with aq
Na2S2O3 and water, dried (Na2SO4), and con-
centrated. The product was purified by silica
gel column chromatography using 2:1 hex-
ane–EtOAc as eluent to give 10 (1.42 g, 58%):
[h]D = +105.6° (c 0.6 CHCl3) 1H NMR
(CDCl3): l 8.07–7.21 (m, 20 H, 4×Ph), 5.84
(d, 1 H, J3,4 2.4 Hz, H-4), 5.74 (dd, 1 H, J1,2
7.9 Hz, J2,3 10.4 Hz, H-2), 5.58 (d, 1 H, J3%4%
3.0 Hz, H-4%), 5.21 (dd, 1 H, H-3), 5.11 (dd, 1
H, J1%2% 8.0 Hz, J2%3% 10.4 Hz, H-2%) 4.77 (d, 1 H,
H-1), 4.72 and 4.40 (each d, 2 H, Jgem 12.2 Hz,
benzylic methylene), 4.42 (d, 1 H, H-1%), 4.14–
4.07 (m, 2 H, H-5, –OCH2CH2–), 3.98 (dd, 1
H, J5,6a 4.7 Hz, J6a,6b 11.0 Hz, H-6a), 3.82 (dd,
1 H, J5,6b 7.3 Hz, H-6b), 3.67–13.56 (m, 4 H,
H-5%, 6%a, 6%b, −OCH2CH2–), 3.50(dd, 1 H,
H-3%), 2.16 and 2.13 (each s, 6 H, 2×OAc),
1.00–0.90 (m, 2 H, –OCH2CH2 –), 0.85 (s, 9
H, 3×CH3), −0.04 (s, 6 H, –Si(CH2)2–),
−0.05(S, 9 H, –Si(CH3)3). Anal. Calcd for
C55H70O16Si2 (1043.3): C, 63.32; H, 6.76.
Found: C, 63.08; H, 6.78.
added AW300 molecular sieves (300 mg) and
the mixture was stirred for 3 h at rt, then
cooled to 0 °C. Boron trifluoride etherate (45
mL) was added, and mixture was stirred for 5 h
at 0 °C then neutralized with Et3N. The solids
were filtered off and washed with CHCl3. The
combined filtrate and washings was succes-
sively washed with water, dried (Na2SO4), and
concentrated. The product was purified by
silica gel column chromatography using 3:2
hexane–EtOAc as eluent to give 8 (46 mg,
1
36%): [h]D+82.5° (c 1.0, CHCl3); H NMR
(CDCl3): l 8.10–7.21 (m, 35 H, 7×Ph), 5.93
(1 H, d, J3%,4% 3.0 Hz, H-4%), 5.91 (d, 1 H, J3,4
3.7 Hz, H-4), 5.77 (dd, 1 H, J1,2 7.9 Hz, J2,3
10.5 Hz, H-2), 5.72 (dd, 1 H, J1%,2% 7.9 Hz, J2%,3%
10.4 Hz, H-2%), 5.57 (dd, 1 H, H-3), 5.51 (dd,
1 H, H-3%), 4.92 (1 H, d, 1 H, H-1%), 4.62 (d, 1
H, H-1), 4.39 (dd, 1 H, J5%,6%a 8.5 Hz, J6%a,6%b
13.5 Hz, H-6%a), 4.25–4.16 (m, 3 H, H-5%,6%b,
–OCH2–), 3.91 (ddd, 1 H, H-5), 3.69 (dd, 1
H, H-6b), 3.17, (dd, 1 H, –OCH2–), 1.34
(br.s, 52 H, 26×CH2), 0.96 (t, 6 H, 2×
–CH2CH3); MALDI-TOFMS: Calcd for
C91H110O18: m/z 1490.8. Found: m/z 1513.7
[M+Na]+, 1529.9 [M+K]+.
2-(Tetradecyl)hexadecyl i-
osyl-(1“6)-i- -galactopyranoside (9).—To a
D
-galactopyran-
D
solution of 8 (40 mg, 27 mmol) in 3:1 MeOH–
1,4-dioxane (2 mL) was added NaOMe (20
mg), and the mixture was stirred for 5 h at rt,
then neutralized with Amberlite IR-120 (H+)
resin. The resin was filtered off and washed
with 1:1 CHCl3–MeOH. The filtrate and
washings were combined and concentrated.
Column chromatography (1:1 CHCl3–MeOH)
of the residue on Sephadex LH-20 gave 9 (20
mg, 94%): [h]D −22.2° (c 0.5, 1:1 CHCl3–
2-(Trimethylsilyl)ethyl 2,4-di-O-acetyl-3-O-
benzyl-i-
D
-galactopyranosyl-(1“6)-2,3,4-tri-
- galactopyranoside (11).—
O - benzoyl - i -
D
Compound 11 (438 mg, 0.42 mmol) was dis-
solved in 3:1:1 AcOH–THF–H2O (50 mL).
After stirring for 3 h at 50 °C, the reaction
mixture was concentrated, and the remaining
solvents were removed by evaporation with
toluene. The product was purified by silica gel
column chromatography using 3:2 hexane–
EtOAc as eluent to give 11 (291 mg, 75%):
[h]D = +95.7° (c 4.1 CHCl3); 1H NMR
(CDCl3): l 8.07–7.21 (m, 20 H, 4×Ph), 5.91
(d, 1 H, J3,4 3.1 Hz, H-4), 5.72 (dd, 1 H, J1,2
7.9 Hz, J2,3 10.4 Hz, H-2), 5.51 (dd, 1 H, H-3),
5.40 (d, 1 H, J3%,4% 3.6 Hz, H-4%), 5.15 (dd, 1 H,
J1%,2% 8.5 Hz, J2%3% 9.8 Hz, H-2%) 4.76 (d, 1 H,
H-1), 4.63 and 4.39 (each d, 2 H, Jgem 12.2 Hz,
benzylic methylene), 4.40 (d, 1 H, H-1%), 4.13–
4.08 (m, 2 H, H-5, –OCH2CH2–), 3.95 (dd, 1
H, J5,6a 5.5 Hz, J6a,6b 10.4 Hz, H-6a), 3 84 (dd,
1 H, J5,6b 6.1 Hz, H-6b), 3.63 (m, 2 H, H-6%a,
–OCH2CH2–), 3.54 (t, 1 H, H-5%), 3.50 (dd, 1
1
MeOH); H NMR (CDCl3–CD3OD): l 4.30
(d, 1 H, J 8.0 Hz, H-la), 4.19 (d, 1 H, J 7.5
Hz, H-lb); MALDI-TOFMS: Calcd for
C42H82O11: m/z 762.6. Found: m/z 785.3 [M+
Na]+, 801.4 [M+K]+.
2-(Trimethylsilyl)ethyl 2,4-di-O-acetyl-3-O-
benzyl-6-O-(tert-butyldimethylsilyl)-i-
D
-gal-
actopyranosy-(1“6)-2,3,4-tri-O-benzoyl-i-
D
-
galactopyranoside (10).—To a solution of 2
(1.39 g, 2.35 mmol) and 4 (1.58 g, 2.82 mmol)
in dry CH2Cl2 (15 mL) was added powdered 4
,
A molecular sieves (3 g), and the mixture was
stirred for 2 h at rt, then cooled to 0 °C. NIS

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
65
gel column chromatography using 15:1 ben-
zene–acetone as eluent to give 14 (127 mg,
89%): [h]D+48.9° (c 0.9, CHCl3); 1H
NMR(CDCl3): l 8.07–7.20 (m, 30 H, 6×Ph),
5.85 (d, 1 H, J3,4 3.0 Hz, H-4), 5.74 (dd, 1 H,
J1,2 7.9 Hz, J2,3 10.3 Hz, H-2), 5.52 (dd, 1 H,
H-3), 5.26–5.22 (m, 1 H, H-2%, 4%), 4.96 (d, 1
H, J1¦,2¦ 3.7 Hz, H-1¦), 4.94–4.60 (m, 6 H,
3×benzylic methylene), 4.77 (d, 1 H, H-1),
4.44 (d, 1H, J1%2% 7.9 Hz, H-1%), 4.14–4.07 (m,
2 H, –CH2CH2Si, H-3¦), 4.02–3.91 (m, 3 H,
H-2¦, 5%, 6a), 3.83–3.79 (m, 5H, H-3%, 4¦, 6b,
6%a, 6%b), 3.67–3.62 (m, 2H, –CH2CH2Si, H-
5¦), 0.85–0.71 (m, 2 H, –CH2CH2Si), −0.08
H, H-3%), 2.25 (br.d, 1 H, OH), 2.17 and 2.05
(each s, 6 H, 2×OAc) 0.95–0.87 (m, 2 H,
–OCH2CH2–), −0.06 (s, 9 H, –Si(CH3)3).
Anal. Calcd for C49H56O16Si (929.1): C, 63.35;
H, 6.08. Found: C, 63.15; H, 6.04.
2-(Trimethylsilyl)ethyl 2,4,6-tri-O-acetyl-i-
D
-galactopyranosyl-(l“6)-2,3,4-tri-O-benz-
oyl-i- -galactopyranoside (12).—To a solu-
D
tion of 11 (104 mg, 0.11 mmol) in pyridine (3
mL) was added Ac2O (2 mL), and the mixture
was stirred for 2 h at rt. At the end of this
time MeOH was added, and the remaining
solvents were removed by evaporation with
toluene. Hydrogenolysis of this residue in
MeOH (4 mL) and AcOH (1 mL) in the
presence of 10% Pd–C (60 mg) was carried
out for 2 h at rt. (Caution: Pd–C and MeOH
in admixture are extremely pyrophoric.) After
removing the Pd–C by filtration, the filtrate
was concentrated. Column chromatography
(10:1 benzene–acetone) of the residue on silica
gel gave 12 (90 mg, 93%): [h]D +90.0° (c 0.6
(s,
9
H, Si(CH3)3). Anal. Calcd for
C71H80O21Si (1297.5): C, 65.73; H, 6.21.
Found: C, 65.52; H, 6.33.
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-h-
L
-fucopyranosy-(1“3)-2,4,6-tri-O-acetyl-i-
-galactopyranosyl-(1“6)-2,3,4-tri-O-benz-
D
oyl-i- -galactopyranoside (15).—A solution
D
of 14 (125 mg, 0.10 mol) in MeOH (5 mL) and
AcOH (0.1 mL) was hydrogenolysed in the
presence of 10% Pd–C (80 mg) for 3 h at
room temperature, then filtered and concen-
trated. The residue was acetylated with Ac2O
(3 mL) in pyridine (4 mL) for 3 h at rt. The
mixture was poured into ice-water and ex-
tracted with CHCl3. The extract was washed
sequentially with 5% HCl, aq NaHCO3 and
water, dried (Na2SO4), and concentrated. The
product was purified by silica gel column
chromatography using 10:1 benzene–acetone
as eluent to give 15 (90 mg, 81%, 2 steps):
[h]D +15.2° (c 0.5, CHCl3); 1H NMR
(CDCl3): l 8.09–7.21 (m, 15 H, 3×Ph), 5.84
(d, 1 H, J3,4 3.0 Hz, H-4), 5.74 (dd, 1 H, J1,2
8.5 Hz, J2,3 10.3 Hz, H-2), 5.52 (dd, 1 H, H-3),
5.29–5.16 (m, 4 H, H-2%, 4%, 3¦, 4¦), 5.26 (d, 1
H, J1¦,2¦ 3.7 Hz, H-1¦), 4.93 (dd, 1 H, H-2¦),
4.77 (d, 1 H, H-1), 4.39 (d, 1 H, J1%,2% 8.6 Hz,
H-1%), 3.93 (dd, 1 H, H-3%), 0.85–0.71 (m, 2 H,
–CH2CH2Si), −0.08 (s, 9 H, Si(CH3)3); Anal.
Calcd for C56H68O24Si (1153.2): C, 58. 32; H,
5. 94. Found: C, 57.91; H, 5.83.
1
CHCl3); H NMR (CDCl3): l 8.07–7.21 (m,
15 H 3×Ph), 5.87 (d, 1 H, J3,4 3.1 Hz, H-4),
5.75 (dd, 1 H, J1,2 7.9 Hz, J2,3 10.4 Hz, H-2),
5.55 (dd, 1 H, H-3), 5.29 (d, 1 H, J3%4% 3.0 Hz,
H-4%), 4.96 (dd, 1 H, J1%2% 7.9 Hz, J2%3% 9.8 Hz,
H-2%) 4.80 (d, 1 H, H-1), 4.51 (d, 1 H, H-1%),
4.19–3.63 (m, 9 H, H-3%, 5, 5%, 6a,6b,6%a,6%b,
–OCH2CH2–), 2.86 (br.s, 1 H, OH), 2.15, 2.13
and 1.97 (each s, 9 H, 3×OAc) 0.98–0.89 (m,
2
H, –OCH2CH2–), −0.05 (s,
9
H,
–Si(CH3)3); Anal. Calcd for C44H52O17Si
(881.0): C, 59.99; H, 5.75. Found: C, 59.82; H,
5.88.
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzyl-h-
L
-fucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i-
D
-galactopyranosyl-(1“6)-2,3,4- tri-O-benz-
oyl-i- -galactopyranoside (14).—To a solu-
D
tion of 12 (97 mg, 0.11 mmol) and 13 (77 mg,
,
0.17 mmol) in CH2Cl2 (2 mL) was added 4 A
molecular sieves (300 mg), and the mixture
was stirred for 2 h at rt, then cooled to 0 °C.
Dimethyl(methylthio)sulfonium triflate (DM-
TST; 130 mg, 0.50 mmol) was added to the
mixture, which was stirred for 5 h at 0 °C,
then neutralized with Et3N. The solids were
filtered off and washed with CHCl3. The com-
bined filtrate and washings were successively
washed with water, dried (Na2SO4), and con-
centrated. The product was purified by silica
2,3,4-Tri-O-acetyl-h-
2,4,6 - tetra - O - acetyl - i -
(1“6)-2,3,4-tri-O-benzoyl-h-
L
-fucopyranosyl-(l“3)-
- galactopyranosyl-
-galactopyran-
D
D
osyl trichloroacetimidate (16).—To a solution
of 15 (81 mg, 0.07 mmol) in CH2Cl2 (3 mL)
cooled to 0 °C, was added CF3COOH (2 mL),

66
N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
mixture was stirred for 5 h at rt, then neutral-
ized with Amberlite IR-120 (H+) resin. The
resin was filtered off and washed with 1:1
CHCl3–MeOH. The filtrate and washings
were combined and concentrated. Column
chromatography (1:1 CHCl3–MeOH) of the
residue on Sephadex LH-20 gave 18 (12 mg,
97%): [h]D −46.3° (c 0.1, 1:1 CHCl3–MeOH);
1H NMR (CDCl3–CD3OD): l 5.16 (d, 1 H, J
4.3 Hz, H-1¦), 4.36 (d, 1 H, J 7.9 Hz, H-1),
4.19 (d, 1 H, J 7.9 Hz, H-1%); MALDI-
TOFMS: Calcd for C48H92O15: m/z 908.6.
Found: m/z 931.4 [M+Na]+, 947.4 [M+
K]+.
and the mixture was stirred for 30 min at rt
and concentrated. Ethyl acetate and toluene
(1:2) were added and then evaporated to give
the 1-hydroxy compound. To a solution of the
residue in CH2Cl2 (0.8 mL) cooled to 0 °C
were added trichloroacetonitrile (210 mL, 2.10
mmol) and DBU (12.7 mL, 84 mmol). The
mixture was stirred for 2 h at 0 °C. After
completion of the reaction, the mixture was
concentrated. Column chromatography (3:2
hexane–EtOAc) of the residue on silica gel
gave 16 (66 mg, 79%) as an amorphous mass:
[h]D+45.8° (c 1.7, CHCl3); 1H NMR
(CDCl3): l 8.61 (s, 1 H, NH), 6.87 (d, 1 H, J1,2
3.7 Hz, H-1), 5.26 (d, 1 H, J1¦,2¦ 3.7 Hz, H-1¦),
4.38 (d, 1 H, J1%,2% 8.6 Hz, H-1%); MALDI-
TOFMS: Calcd for C53H56Cl3NO24: m/z
1196.7. Found: m/z 1219.5 [M+Na]+, 1235.9
[M+K]+.
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
oyl - i -
acetyl-3-O-benzyl-i-
2,3,4 - tri - O - benzoyl - i -
D
- galactopyranosyl - (1“6) - 2,4 - di-O-
D
-galactopyranosyl-(1“6)-
- galactopyranoside
D
(19).—To a solution of 11 (108 mg, 0.12
mmol) and 5 (120 mg, 0.17 mmol) in dry
2-(Tetradecyl)hexadecyl 2,3,4-tri-O-acetyl-
,
CH2Cl2 (2 mL) was added powdered 4 A
h -
acetyl-i-
O-benzoyl-i-
L
- fucopyranosyl - (1“3) - 2,4,6 - tetra - O-
-galactopyranosyl-(1“6)-2,3,4-tri-
-galactopyranoside (17).—To a
molecular sieves (300 mg), and the mixture
was stirred for 2 h at rt, then cooled to 0 °C.
NIS (57 mg, 0.25 mmol) and TfOH (3 mL, 35
mmol) were added to the mixture, which was
stirred for 20 min at 0 °C, then neutralized
with Et3N. The solids were filtered off and
washed with CHCl3. The combined filtrate
and washings were successively washed with
aq Na2S2O3 and water, dried (Na2SO4), and
concentrated. The product was purified by
silica gel column chromatography using 2:1
hexane–EtOAc as eluent to give 19 (128 mg,
D
D
solution of the trichloroacetimidate 16 (66 mg,
55 mmol) and 2-(tetradecyl)hexadecanol (36
mg, 83 mmol) in CH2Cl2 (1 mL) were added
AW300 molecular sieves (300 mg) and the
mixture was stirred for 3 h at rt, then cooled
to 0 °C. Boron trifluoride etherate (30 mL) was
added, and mixture was stirred for 5 h at 0 °C
then neutralized with Et3N. The solids were
filtered off and washed with CHCl3. The com-
bined filtrate and washings was successively
washed with water, dried (Na2SO4), and con-
centrated. The product was purified by silica
gel column chromatography using 3:2 hex-
ane–EtOAc as eluent to give 17 (46 mg, 37%):
[h]D+15.2° (c 0.2, CHCl3); 1H NMR
(CDCl3): l 5.25 (d, 1 H, J1¦,2¦ 3.7 Hz, H-1¦),
4.69 (d, 1 H, J1,2 8.0 Hz, H-1), 4.38 (d, 1 H,
J1%,2% 7.9 Hz, H-1%), 3.92 (dd, 1 H, –OCH2–),
3.34 (dd, 1H, –OCH2–), 1.34 (br.s, 52 H,
26×CH2), 0.96 (t, 6 H, 2× –CH2CH3);
MALDI-TOFMS: Calcd for C81H116O24: m/z
1472.8. Found: m/z 1495.6 [M+Na]+, 1511.8
[M+K]+.
1
78%): [h]D +84.5° (c 1.0, CHCl3); H NMR
(CDCl3): d 8.12–7.20 (m, 40H, 8×Ph), 5.99
(d, 1 H, J3¦,4¦ 3.7 Hz, H-4¦), 5.82 (d, 1 H, J3,4
3.0 Hz, H-4), 5.77–5.72 (m, 2 H, H-2, 2¦),
5.61 (dd, 1 H, J2¦,3¦ 10.3 Hz, H-3¦), 5.53 (d, 1
H, J3%,4% 3.0 Hz, H-4%), 5.52 (dd, 1 H, H-3), 5.07
(dd, 1 H, J1%,2% 7.9 Hz, J2%,3% 9.8 Hz, H-2%), 4.84
(d, 1 H, J1¦,2¦ 7.9 Hz, H-1¦), 4.76 (d, 1 H, H-1),
4.67 (dd, 1 H, J5¦,6¦a 6.7 Hz, J6¦a,6¦b 11.0 Hz,
H-6¦a), 4.61 and 4.26 (each d, 2 H, Jgem 12.2
Hz, benzylic methylene) 4.42 (dd, 1 H, J5¦,6¦
6.7 Hz, H-6¦), 4.33 (d, 1 H, H-1%), 4.32 (t, 1 H,
H-5¦), 4.25–4.07 (m, 2 H, H-5, –CH2CH2Si),
3.95–3.60 (m, 6 H, H-5%, 6a, 6b, 6%a, 6%b,
–CH2CH2Si), 3.37 (dd, 1 H, H-3%), 2.09 and
2.01 (each s, 6 H, 2×OAc), 0.95–0.85 (m, –2
H, CH2CH2Si), −0.08 (9 H, s, Si(CH3)3).
Anal. Calcd for C83H82O25Si (1507.7): C,
66.12; H, 5.48. Found: C, 65.92; H, 5.35.
2-(Tetradecyl)hexadecyl h-
L
-fucopyranosyl-
(1“3) - i - - galactopyranosyl - (1“6) - i - -
D
D
galactopyranoside (18).—To a solution of 17
(20 mg, 13 mmol) in 3:1 MeOH–1,4-dioxane
(2 mL) was added NaOMe (20 mg), and the

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
67
CH2Cl2 (4 mL), cooled to 0 °C, was added
CF3COOH (3 mL), and the mixture was
stirred for 30 min at rt and concentrated.
Ethyl acetate and toluene (1:2) were added
and then evaporated to give the 1-hydroxy
compound. To a solution of the residue in
CH2Cl2 (2 mL) cooled at 0 °C were added
trichloroacetonitrile (380 mL, 3.80 mmol) and
DBU (23 mL, 152 mmol). The mixture
was stirred for 2 h at 0 °C. After completion
of the reaction, the mixture was concen-
trated. Column chromatography of the
residue on silica gel (3:2 hexane–EtOAc) gave
22 (105 mg, 55%) as an amorphous mass:
[h]D +61.4° (c 1.3, CHCl3); 1H NMR
(CDCl3): l 8.63 (s, 1 H, NH), 6.85 (d, 1 H, J
3.7 Hz, H-1), 4.74 (d, 1 H, J 7.9 Hz, H-1¦),
4.41 (d, 1 H, J 7.9 Hz, H-1%); MALDI-
TOFMS: Calcd for C75H66Cl3NO26: m/z
1502.7. Found: m/z 1525.6 [M+Na]+, 1541.8
[M+K]+.
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
oyl-i- -galactopyranosyl-(1“6)-2,4-di-O-
acetyl-i- -galactopyranosyl-(1“6)-2,3,4-tri-
O-benzoyl-i- -galactopyranoside (20).—A
D
D
D
solution of 19 (367 mg, 0.24 mol) in MeOH (8
mL) and AcOH (2 mL) was hydrogenolysed
in the presence of 10% Pd–C (200 mg) for 18
h at rt, then filtered and concentrated. (Cau-
tion: Pd–C and MeOH in admixture are
extremely pyrophoric.) The product was
purified by silica gel column chromatography
using 3:2 hexane–ethyl acetate as eluent to
give 20 (284 mg, 82%) [h]D +97.8° (c 1.5,
1
CHCl3); H NMR (CDCl3): l 8.10–7.21 (m,
35 H, 7×Ph), 5.98 (d, 1 H, J3¦,4¦ 3.0 Hz,
H-4¦), 5.83 (d, 1 H, J3,4 3.7 Hz, H-4), 5.76–
5.70 (m, 2 H, H-2, 2¦), 5.59 (dd, 1 H, J2¦,3¦
10.4 Hz, H-3¦), 5.53 (dd, 1 H, H-3), 5.29 (d, 1
H, J3%,4% 3.0 Hz, H-4%), 4.89 (dd, 1 H, J1%,2% 7.9
Hz, J2%,3% 10.4 Hz, H-2%), 4.83 (d, 1 H, H-1¦),
4.82 (d, 1 H, H-1), 4.64 (dd, 1 H, J5¦,6¦a 6.7 Hz,
J6¦a,6¦b 11.7 Hz, H-6¦a), 4.42 (dd, 1 H, J5¦,6¦ 6.7
Hz, H-6¦), 4.38 (d, 1 H, H-1%), 4.30 (t, 1 H,
H-5¦), 4.13–4.07 (m, 2 H, H-5, –CH2CH2Si),
3.95–3.58 (m, 7 H, H-3%, 5%, 6a, 6b, 6%a, 6%b,
–CH2CH2Si), 2.52 (d, 1 H, J 6.1 Hz, OH),
2.02 and 2.01 (each s, 6 H, 2×OAc), 0.96–
0.87 (m, 2 H, –CH2CH2Si), −0.08 (s, 9 H,
Si(CH3)3). Anal. Calcd for C76H76025Si
(1417.5): C, 64.40; H, 5.40. Found: C, 63.97;
H, 5.45.
2-(Tetradecyl)hexadecyl
benzoyl-i- -galactopyranosyl-(1“6)-2,3,4-
tri - O - acetyl - i - - galactopyranosyl - (l“6)-
2,3,4 - tri - O - benzoyl - i - - galactopyranoside
2,3,4,6-tetra-O-
D
D
D
(23).—To a solution of the trichloroacetimi-
date 22 (50 mg, 33 mmol) and 2-(tetradec-
yl)hexadecanol (30 mg, 68 mmol) in CH2Cl2 (1
mL) were added AW300 molecular sieves (300
mg), and the mixture was stirred for 3 h at rt,
then cooled to 0 °C. Boron trifluoride etherate
(19 mL) was added, and the mixture was
stirred for 5 h at 0 °C then neutralized with
Et3N. The solids were filtered off and washed
with CHCl3. The combined filtrate and wash-
ings was successively washed with water, dried
(Na2SO4), and concentrated. The product was
purified by silica gel column chromatography
using 3:2 hexane–EtOAc as eluent to give 23
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
oyl - i -
O-acetyl-i-
tri-O-benzoyl-i-
D
- galactopyranosyl - (1“6) - 2,3,4 - tri-
-galactopyranosyl-(1“6)-2,3,4-
-galactopyranoside (21).—
D
D
Compound 20 (100 mg, 0.07 mmol) was acetyl-
ated with Ac2O (2 mL) in pyridine (3 mL) for
3 h at rt. Workup as described for 15 gave 21
1
(98 mg, 96%): [h]D +84.5° (c 1.2, CHCl3); H
1
NMR (CDCl3): l 4.81 (d, 1 H, J1¦,2¦ 7.3 Hz,
H-1¦), 4.80 (d, 1 H, J1,2 8.0 Hz, H-1), 4.38 (d,
1 H, J1%,2% 8.0 Hz, H-1%), 2.08, 2.06 and 1.96
(each s, 9 H, 3×OAc). Anal. Calcd for
C78H78O26Si (1459.6): C, 64.19; H, 5.39.
Found: C, 64.02; H, 5.42.
(46 mg, 49%): [h]D +64.4° (c 0.8, CHCl3); H
NMR (CDCl3): l 4.77 (d, 1 H, J1¦,2¦ 7.4 Hz,
H-1¦), 4.70 (d, 1 H, J1,2 7.9 Hz, H-1), 4.43 (d,
1 H, J1%,2%, 7.9 Hz, H-1%), 3.92 (dd, 1 H, –
OCH2–), 3.33 (dd, 1 H, –OCH2–), 1.34 (br.s,
52 H, 26×CH2), 0.96 (t, 6 H, 2× –CH2CH3);
MALDI-TOFMS: Calcd for C103H126O26: m/z
1778.8. Found: m/z 1801.7 [M+Na]+, 1817.7
[M+K]+.
2,3,4,6-Tetra-O-benzoyl-i-
osyl-(1“6)-2,3,4-tri-O-acetyl-h-
pyranosyl - (1“6) - 2,3,4 - tri - O - benzoyl - h-
D
-galactopyran-
D
-galacto-
D
-
galactopyranosyl trichloroacetimidate (22).—
To a solution of 21 (185 mg, 0.16 mmol) in
2-(Tetradecyl)hexodecyl i-
D
-galactopyran-
osyl - (1“6) - i - - galactopyranosyl - (1“6)-
D

68
N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
i-
D
-galactopyranoside (24).—To a solution
was hydrogenolysed in the presence of 10%
Pd–C (200 mg) for 18 h at rt, then filtered
and concentrated. The residue was acetylated
with Ac2O (3 mL) in pyridine (4 mL) for 3
h at rt. Workup was carried out as described
for 15 and the product was purified by silica
gel column chromatography using 1:1 hex-
ane–EtOAc as an eluent to give 26 (235 mg,
92%, 2 steps): [h]D +42.3° (c 1.7, CHCl3);
1H NMR (CDCl3): l 5.22 (d, 1 H, J 3.7 Hz,
H-1 of Fuc), 4.79 (d, 1 H, J 7.9 Hz, H-1c),
4.76 (d, 1 H, J 7.9 Hz, H-la), 4.26 (d, 1 H,
of 23 (29 mg, 16.3 mmol) in 3:1 MeOH–1,4-
dioxane (2 mL) was added NaOMe (20 mg),
and the mixture was stirred for 5 h at rt,
then neutralized with Amberlite IR-120 (H+)
resin. The resin was filtered off and washed
with 1:1 CHCl3–MeOH. The filtrate and
washings were combined and concentrated.
Column chromatography (1:1 CHCl3–
MeOH) of the residue on Sephadex LH-20
gave 24 (14 mg, 93%): [h]D −23.8° (c 0.2,
1:1 CHCl3–MeOH); 1H NMR (CDCl3–
CD3OD): l 4.30 (d, 1 H, J 8.0 Hz, H-1),
4.19 (d, 1 H, J 7.5 Hz, H-1%), 4.10 (d, 1 H,
J 7.5 Hz, H-1¦); MALDI-TOFMS: Calcd for
C48H92O16: m/z 924.6. Found: m/z 947.3
[M+Na]+, 963.5 [M+K]+.
J
8.0 Hz, H-1b). Anal. Calcd for
C88H92O32Si (1689.8): C, 62.55; H, 5.49.
Found: C, 62.25; H, 5.46.
2,3,4,6-Tetra-O-benzoyl-i-
osyl-(1“6)-[2,3,4-tri-O-benzyl-h-
osyl-(1“3)]-[2,4-di-O-acetyl-i-
anosyl - (1“6) - 2,3,4 - tri - O - benzoyl - i -
D
-galactopyran-
-fucopyran-
-galactopyr-
L
D
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
D
-
oyl-i-
O-benzyl-h-
acetyl-i- -galactopyranosyl-(1“6)-2,3,4-tri-
O-benzoyl-i- -galactopyranoside (25).—To a
solution of 20 (180 mg, 0.13 mmol) and 13
(88 mg, 0.19 mmol) in CH2Cl2 (2 mL) was
D
-galactopyranosyl-(1“6)-[2,3,4-tri-
galactopyranosyl trichloroacetimidate (27).—
To a solution of 26 (165 mg, 98 mmol) in
CH2Cl2 (3 mL) cooled to 0 °C was added
CF3COOH (2 mL), and the mixture was
stirred for 30 min at rt and concentrated.
Ethyl acetate and toluene (1:2) were added
and then removed to give the 1-hydroxy
compound. To a solution of the residue in
CH2Cl2 (2 mL) cooled to 0 °C were added
trichloroacetonitrile (300 mL, 3 mmol) and
DBU (18 mL, 0.12 mmol). The mixture was
stirred for 2 h at 0 °C. After completion of
the reaction, the mixture was concentrated.
Column chromatography (20:1 benzene–ace-
tone) of the residue on silica gel gave 27
(162 mg, 95%) as an amorphous mass:
[h]D +45.8° (c 0.2, CHCl3); 1H NMR
(CDCl3): l 8.61 (s, 1H, NH), 6.85 (d, 1 H, J
3.7 Hz, H-1a), 5.20 (d, 1 H, J 3.7 Hz, H-1
of Fuc), 4.73 (d, 1 H, J 7.9 Hz, H-1c), 4.24
(d, 1 H, J 7.9 Hz, H-1b); MALDI-TOFMS:
Calcd for C85H80Cl3NO32: m/z 1732.8.
Found: m/z 1755.9 [M+Na]+, 1771.8 [M+
K]+.
L
-fucopyranosyl-(1“3)]-2,4,-di-O-
D
D
,
added 4 A molecular sieves (300 mg), and
the mixture was stirred for 2 h at rt, then
cooled to 0 °C. DMTST (147 mg, 0.57
mmol) was added to the mixture, which was
stirred for 5 h at 0 °C, then neutralized with
Et3N. The solids were filtered off and
washed with CHCl3. The combined filtrate
and washings were successively washed with
water, dried (Na2SO4), and concentrated.
The product was purified by silica gel
column chromatography using 15:1 benz-
ene–acetone as eluent to give 25 (209 mg,
1
90%): [h]D+57.2° (c 1.3, CHCl3); H NMR
(CDCl3): l 4.96 (d, 1 H, J 3.7 Hz, H-1 of
Fuc), 4.79 (d, 1 H, J 7.9 Hz, H-1a), 4.79 (d,
1 H, J 7.9 Hz, H-1c), 4.33 (d, 1 H, J 7.9
Hz, H-1b). Anal. Calcd for C103H104O29Si
(1834.1): C, 67.45; H, 5.72. Found: C, 67.20;
H, 5.77.
2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-benz-
2-(Tetradecyl)hexadecyl
benzoyl-i- -galactopyranosyl-(l“6)-[2,3,4-
tri-O-acetyl-h- -fucopyranosyl-(1“3)]-2,4,-
di-O-benzoyl-i- -galactopyranosyl-(1“6)-
2,3,4 - tri - O - benzoyl - i - - galactopyranoside
2,3,4,6-tetra-O-
oyl-i-
O-acetyl-h-
acetyl-i- -galactopyranosyl-(1“6)-2,3,4-tri-
O-benzoyl-i- -galactopyranoside (26).—A
solution of 25 (278 mg, 0.17 mol) in MeOH
(4 mL), THF (1 mL) and AcOH (0.1 mL)
D
-galactopyranosyl-(1“6)-[2,3,4-tri-
D
L
-fucopyranosyl-(1“3)]-2,4,-di-O-
L
D
D
D
D
(28).—(a) To a solution of the trichloroace-
timidate 27 (30 mg, 17 mmol) and 2-(tetradec-

N. Hada et al. / Carbohydrate Research 316 (1999) 58–70
69
Acknowledgements
yl)hexadecanol (15 mg, 34 mmol) in CH2Cl2
(1 mL) were added AW300 molecular sieves
(300 mg), and the mixture was stirred for 3
h at rt, then cooled to 0 °C. Boron trifl-
uoride etherate (25 mL) was added, and the
mixture was stirred for 5 h at 0 °C, then
neutralized with Et3N. The solids were
filtered off and washed with CHCl3. The
combined filtrate and washings was succes-
sively washed with water, dried (Na2SO4),
and concentrated. The product was purified
by silica gel column chromatography using
8:1 benzene–acetone as an eluent to give 28
(10 mg, 29%). (b) To a solution of the
trichloroacetimidate 27 (95 mg, 55 mmol)
and 2-(tetradecyl)hexadecanol (48 mg, 110
The authors would like to thank Miss T.
Naito for performing the microanalyses at
Nagoya City University, Nagoya, Japan.
This work was supported by the Sasagawa
Scientific Research Grant from The Japan
Science Society and also supported by a
Grant-in-Aid for Encouragement of Young
Scientists, No. 70296531, from the Ministry
of Education, Science, Sports and Culture of
Japan.
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