Synthetic studies on glycosphingolipids from Protostomia phyla: Total syntheses of glycosphingolipids from the parasite, Echinococcus multilocularis

Article abstract of DOI:10.1016/j.carres.2004.09.015

Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with be

Full text of DOI:10.1016/j.carres.2004.09.015

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2749–2759
Synthetic studies on glycosphingolipids from Protostomia phyla:
total syntheses of glycosphingolipids from the parasite,
Echinococcus multilocularis
Takeshi Yamamura,a Noriyasu Hada,a Asuka Kaburaki,a
Kimiaki Yamanob and Tadahiro Takedaa,*
aKyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
bDepartment of Biology, Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan
Received 6 July 2004; accepted 21 September 2004
Abstract—Efficient and systematic syntheses of four neutral glycosphingolipids that have been isolated from the metacestodes of
Echinococcus multilocularis have been achieved. A key step is the direct glycosylation of galactosyl donors using thioglycosides with
benzoyl ceramide in the presence of N-iodosuccinimide (NIS)/TfOH, which gave the desired oligosaccharide derivatives. The fully
protected glycosides 13, 20, 22 and 25 were deprotected to give four target glycosphingolipids (1–4).
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Glycosphingolipids; Echinococcus multilocularis; Chemical synthesis; Benzoyl ceramide
1. Introduction
In one of our previous papers we reported1 the synthesis
of four glycosphingolipid analogues containing
2-branched fatty alkyl residues in place of ceramide.
Therefore, at this time we report the total synthesis of
four glycosphingolipids. Excellent pioneering studies of
the coupling of oligosaccharides with sphingolipids have
been reported by Ogawa,8–11 Schmidt12–15 and Hase-
gawa.16–19 Reactions to couple the oligosaccharide and
the sphingolipid employ a glycosyl imidate as the donor
and an azidosphingosine as the acceptor in the presence
of TMSOTf or BF3OEt2.12–19 The coupling with azi-
dosphingosine is now popularly employed because of
the good yield for coupling and the ability to accept a
series of fatty acids as components of ceramide,
although the resulting glycosyl azidosphingosine neces-
sitates further transformation. Furthermore, the cou-
In our continuing studies to elucidate the mechanism of
host–parasite interaction, we have synthesized glyco-
sphingolipids from various parasites.1–4 Persat et al.5–7
found some novel glycosphingolipids, b-D-Galp-(1!6)-
b-D-Galp-(1!1)-Cer (1), a-L-Fucp-(1!3)-b-D-Galp-
(1!6)-b-D-Galp-(1!1)-Cer (2), b-D-Galp-(1!6)-b-D-
Galp-(1!6)-b-D-Galp-(1!1)-Cer (3) and b-D-Galp-
(1!6)-[a-L-Fucp-(1!3)]-b-D-Galp-(1!6)-b-D-Galp-
(1!1)-Cer (4) in metacestodes of the parasite, Echino-
coccus multilocularis, which belongs to the class Ces-
toda. It is particularly interesting to note that these
glycosphingolipids contain the unique structures of a
b-D-Galp-(1!6)-b-D-Galp-core and a fucose residue
and point to the functional importance of glycolipids
in parasitism that are recognized by serum antibodies
from different patients with alveolar hydatid disease.
pling of glycosyl fluorides as
a
donor with
sphingolipids as an acceptor have been also reported
by Ogawa11 and Nicolaou.20 However, no one has
reported the coupling of a thioglycoside with sphingo-
lipids except the instance with azidosphingosine by
Wong21 and Danishefsky.22 We synthesized the four
*
Corresponding author. Tel.: +81 03 5400 2696; fax: +81 03 5400
2556; e-mail: takeda-td@kyoritsu-ph.ac.jp
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.09.015

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T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
target glycolipids 1–4, as a systematic, integrated
approach for the synthesis of tetrasaccharides using
thioglycoside derivatives at the reducing end of the
oligosaccharide moiety. This is the first report of the
glycosylation of a thioglycoside with sphingolipids.
subsequent chloroacetylation gave compound 7. For
selective removal of the 2-(trimethylsilyl)ethyl (SE)
group, 7 was treated23 with trifluoroacetic acid in dichlo-
romethane to give the 1-hydroxy compound, which, on
further treatment24 with trichloroacetonitrile in the
OH
OH
OH
OH
O
O
O
HO
O
O
OH
OH
OH
OH
O
H3C
O
O
Cer
HO
OH
Cer
HO
OH
HO
OH
OH
2
1
OH
HO
OH
HO
OH
OH
O
O
O
O
OH
OH
OH
OH
O
O
O
O
HO
O
OH
OH
OHOH
HO
H3C
O
O
O
OH
Cer
Cer
HO
HO
OH
OH
OH
3
4
2. Results and discussion
2.1. Syntheses of monosaccharide derivatives
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
in dichloromethane, gave only the corresponding a-tri-
chloroacetimidate 8.
Syntheses of the additional galactopyranosyl building
blocks 8 were carried out as depicted in Scheme 1. 2-
(Trimethylsilyl)ethyl 2,4-di-O-benzoyl-3-O-benzyl-6-O-
levulinoyl-b-D-galactopyranoside (6) was prepared from
known 2-(trimethylsilyl)ethyl b-D-galactopyranoside
(5)23 by regioselective benzylation of the in situ prepared
stannylidene derivative with benzyl bromide and subse-
quent regioselective levulinoylation with levulinic acid,
followed by benzoylation. Removal of the benzyl groups
from 6 by catalytic hydrogenolysis over 10% Pd–C and
2.2. Systematic syntheses of the each
of the glycosphingolipids
In this work, four kinds of target glycolipids 1–4 were
synthesized by stepwise condensation of suitably pro-
tected monosaccharide units. A systematic plan for syn-
thesis of these compounds was designed as shown in
Schemes 1–5.
First, we chose compounds 11, 17, 19 and 24, which
were phenyl thioglycoside derivatives, as the intermedi-
OH
HO
OH
OBz
OLev
O
O
a
O
O
TMS
BnO
TMS
OH
OBz
5
6
OBz
OBz
OLev
O
OLev
b
O
c
O
ClAcO
ClAcO
TMS
OBz
BzO
O
CCl3
NH
7
8
Scheme 1. Reagents: (a) (i) Bu2SnO, benzene, Bu4NBr, BnBr, benzene; (ii) DCC, DMAP, LevOH, CH2Cl2; (iii) BzCl, Pyr., 71% three steps; (b) (i)
H2, Pd–C, AcOH, 2:1 THF–MeOH; (ii) ClCH2COCl, Pyr., CH2Cl2, 88%; (c) (i) CF3COOH, CH2Cl2; (ii) CCl3CN, DBU, CH2Cl2, 74%.

T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
2751
OBz
OBz
O
OBz
BzO
OBz
OBz
O
O
CCl3
NH
9
BzO
O
+
OBz
OBz
a
OBz
OH
O
O
SPh
BzO
SPh
BzO
OBz
OBz
11
10
NHCOC15H31
C15H31
OR
RO
OR
HO
O
O
OBz
OR
OR
12
b
NHCOC15H31
C15H31
O
O
RO
OR
OR
13 R = Bz
1 R = H
c
Scheme 2. Reagents: (a) TMSOTf, CH2Cl2, 91%; (b) NIS, TfOH, CH2Cl2, 44%; (c) NaOMe, 1:1 dioxane–MeOH, quant.
OBz
OLev
O
OBz
OLev
O
a
b
O
O
ClAcO
8 + 10
HO
OBz
BzO
BzO
14
BzO OBz
O
O
SPh
SPh
BzO
15
OBz
OBz
c
OBz
OBz
O
OBz
OBz
O
BzO
OBz
OH
BzO
O
BzO
O
O
CCl3
NH
BzO
OBz
9
O
ClAcO
O
BzO OBz
O
O
ClAcO
O
d
BzO
OBz
SPh
BzO
OBz
SPh
BzO
16
17
OBz
Scheme 3. Reagents: (a) TMSOTf, CH2Cl2, quant.; (b) thiourea, 6:1 pyr.–EtOH, 75%; (c) hydrazine acetate, 10:1 THF–MeOH, 88%; (d) TMSOTf,
CH2Cl2, 92%.
ates. Glycosylation of 1025 with 926 in the presence of
trimethylsilyl triflate TMSOTf (cat.)27 and 4A MS in
dichloromethane gave the desired disaccharide 11
urea and 6:1 pyridine–ethanol gave the partially
protected disaccharide derivative 15. Glycosylation of
15 with phenyl 2,3,4-tri-O-benzyl-1-thio-b-L-fucopyr-
anoside 1828 in the presence of NIS and TfOH29 as
the glycosyl promoter and 4A MS in dichloromethane
gave the desired a-glycoside 19 in 72% yield.
1
(91%) as evidenced by H NMR spectroscopy (H-10
4.91ppm, J 7.9Hz). For the other targets, the common
donor 8 was chosen for compounds 17, 19 and 24, which
were condensed with 10 under the agency of TMSOTf
(cat) to yield disaccharide 14 (quant) as evidenced by
1H NMR spectroscopy (H-10 4.42ppm, J 8.0Hz). Selec-
tive removal of the chloroacetyl group in 14 with thio-
1
Significant signals of the fucose unit in the H NMR
spectrum showed a signal for the anomeric hydrogen
atoms at d 5.05 (d, J 2.0Hz). The a-L configuration of
the newly formed glycosidic bond was also indicated

2752
T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
SPh
OBn
Me
O
OBz
OLev
O
OBn
O
BnO
O
18
a
BzO
OBz
BzO
19
Me
O
O
OBn
15
SPh
OBn
BnO
OBz
NHCOC15H31
C15H31
OR2
OR3
O
HO
OBz
12
O
O
R2O
OR2
R2O
b
Me
R1O
O
NHCOC15H31
C15H31
OR1
O
O
OR1
OR2
OR2
20 R1 = Bn, R2 = Bz, R3 = Lev
21 R1 = H, R2 = Bz, R3 = Lev
2 R1 = H, R2 = H, R3 = H
c
d
Scheme 4. Reagents: (a) NIS, TfOH, CH2Cl2, 72%; (b) NIS, TfOH, CH2Cl2, 54%; (c) H2, Pd–C, 2:1 THF–MeOH, 84%; (d) NaOMe, 1:1 dioxane–
MeOH, 73%.
OR1
OR1
O
NHCOC15H31
C15H31
HO
R1O
O
R1O
1
OBz
OR
R2O
12
O
a
O
17
R1O
1
OR
R1O
NHCOC15H31
C15H31
O
O
OR1
c
OBz
OBz
O
OR1
BzO
O
BzO
OBz
22 R1 = Bz, R2 = ClAc
3 R1 = H, R2 = H
O
b
O
HO
BzO
OBz
O
SPh
BzO
23
OBz
Me
SPh
OBn
O
d
OBn
18
BnO
OR2
OR2
O
OBz
OBz
O
R2O
O
NHCOC15H31
C15H31
R2O
2
BzO
O
OR
HO
BzO
OBz
O
O
O
OBz
12
O
O
2
O
R2O
e
OR
R2O
NHCOC15H31
C15H31
Me
BzO
O
OR1
O
OR1
OBz
Me
O
O
O
OBn
24
SPh
R1O
OR2
BzO
OR2
OBn
BnO
OBz
25 R1 = Bn, R2 = Bz,
26 R1 = H, R2 = Bz,
4 R1 = H, R2 = H,
f
g
Scheme 5. Reagents: (a) NIS, TfOH, CH2Cl2, 33%; (b) NaOMe, 1:1 dioxane–MeOH, quant.; (c) thiourea, 6:1 Pyr.–EtOH, 84%; (d) NIS, TfOH,
CH2Cl2, 91%; (e) NIS, TfOH, CH2Cl2, 27%; (f) H2, Pd–C, 2:1 THF–MeOH, 98%; (g) NaOMe, 1:1 dioxane–MeOH, 94%.

T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
2753
by the JC,H value30 of 169.7Hz in the 13C NMR
spectrum.
3.2. 2-(Trimethylsilyl)ethyl 2,4-di-O-benzoyl-3-O-benzyl-
6-O-levulinoyl-b-D-galactopyranoside (6)
Compound 14 was converted by O-delevulinoylation
using hydrazine acetate and condensed with 9 in the
presence of TMSOTf as described for compound 11 to
give the trisaccharide 17 (92%), as evidenced by 1H
NMR spectroscopy (H-100 4.63ppm, J 7.9Hz). Removal
of the 30-O-chloroacetyl group from 17 by thiourea gave
the acceptor 23 (84%) for the tetrasaccharide derivative.
Furthermore the glycosylation of 23 with 18 in the pres-
ence of NIS/TfOH as described for 19 gave the tetrasac-
A mixture of 2-(trimethylsilyl)ethyl b-D-galactopyrano-
side (5) (2.00g, 7.13mmol), dibutyltin oxide (1.93g,
7.75mmol) and 150mL of dry benzene was stirred under
reflux for 5h. Benzene (75mL) was distilled off, and the
solution was cooled to 60ꢁC and treated with Bu4NBr
(2.86g, 8.87mmol) and BnBr (1.73mL, 14.7mmol).
The reaction mixture was stirred for 4.5h, and the solu-
tion was concentrated. Purification of the residue by col-
umn chromatography (20:1 CHCl3–MeOH) on silica gel
gave 2-(trimethylsilyl)ethyl 3-O-benzyl-b-D-galactopyr-
anoside (2.60g, quant). To a solution of this compound
(201mg, 0.54mmol) in CH2Cl2 (2mL) was added levuli-
nic acid (61lL, 0.60mmol), DCC (202mg, 0.98mmol)
and DMAP (20mg, 0.16mmol). The reaction mixture
was stirred for 3h at rt, then extracted with CHCl3,
washed with 5% HCl, dried (MgSO4), and concentrated.
The crude product was purified by silica gel column
chromatography using 35:1 CHCl3–MeOH as eluent to
give 2-(trimethylsilyl)ethyl 3-O-benzyl-6-O-levulinoyl-b-
D-galactopyranoside (223mg, 87.7%). To a solution of
this compound (223mg, 0.48mmol) in pyridine (2mL)
was added benzoyl chloride (0.22mL, 1.90mmol), and
the mixture was stirred for 2h, then extracted with
CHCl3, washed with 5% HCl, aq NaHCO3 and water,
dried (MgSO4) and concentrated. The product was puri-
fied by silica gel column chromatography using 20:1 tol-
uene–acetone as eluent to give compound 6 (264mg,
1
charide derivative 24 (91%), as evidenced by H NMR
spectroscopy (H-1 of Fuc 5.06ppm, J 3.7Hz).
Next, compounds 11, 17, 19 and 24 were used as gly-
cosyl donors without further conversion. Glycosylation
of benzoylceramide 1231 with the each glycosyl donors,
which was carried out in the presence of NIS/TfOH
and 4A MS, afforded the desired b-glycosides 13
(44%), 20 (54%), 22 (33%) and 25 (27%). Removal of
the benzyl groups from 20 and 25 by catalytic hydro-
genolysis over 10% Pd–C in THF–MeOH gave 21 and
26, and subsequent deacylation of 13, 21, 22 and 26 with
sodium methoxide in 1:1 methanol–1,4-dioxane afforded
the desired target glycolipids 1–4.
In conclusion, a stereocontrolled, efficient route to the
Echinococcus glycosphingolipids was exploited by use of
phenyl thioglycosides as key glycosyl donors.
Biological testing results from studies of the sera from
patients with alveolar hydatid disease for these com-
pounds will be reported in detail elsewhere.
24
1
82.0%). ½aꢀ +83.5 (c 0.8, CHCl3); H NMR (CDCl3): d
D
8.24–7.14 (m, 15H, Ph·3), 5.92 (d, 1H, J3,4 3.1Hz, H-
4), 5.57 (dd, 1H J1,2 7.9Hz, J2,3 9.8Hz, H-2), 4.77 and
4.60 (each d, 2H, Jgem 12.8Hz, benzyl methylene), 4.68
(d, 1H, H-1), 4.44 (dd, 1H, J5,6a 6.7Hz, J6a,6b 11.0Hz,
H-6a), 4.27 (dd, 1H, J5,6b 6.7Hz, H-6b), 4.11–4.06 (m,
1H, –OCH2CH2–), 4.03 (t, 1H, H-5), 3.85 (dd, 1H, H-
3), 3.68–3.62 (m, 1H, –OCH2CH2–), 2.85 (t, 2H,
–OCOCH2CH2COCH3), 2.65 (t, 2H, –OCOCH2CH2-
COCH3), 2.29 (s, 3H, –OCOCH2CH2COCH3), 1.02–
0.93 (m, 2H, –OCH2CH2–), ꢁ0.09 (s, 9H, –Si(CH3)3);
13C NMR (CDCl3): d 206.6, 172.3, 165.9, 137.3, 133.3,
130.1, 129.9, 129.4, 128.5, 128.2, 128.0, 127.6, 101.0,
76.1, 71.2, 70.9, 70.7, 67.5, 66.4, 62.1, 38.0, 29.8, 27.9,
17.9. MALDI-TOFMS: Calcd for C37H44O10Si: m/z
676. Found: m/z 699 [M+Na]+. HRFABMS: Calcd for
C37H44O10SiNa [M+Na]+: m/z 699.2601. Found: m/z
699.2648.
3. Experimental
3.1. General
Optical rotations were determined with a Jasco digital
1
polarimeter. H NMR and 13C NMR spectra were re-
corded with a JNM A 500 FT NMR spectrometer with
Me4Si as the internal standard for solutions in CDCl3.
MALDI-TOFMS was recorded on a Perceptive Voyager
RP mass spectrometer. High-resolution mass spectra
were recorded on a JEOL JMS-700 under FAB condi-
tions. TLC was performed on Silica Gel 60-F254 (E.
Merck) with detection by quenching of UV fluorescence
and by spraying with 10% H2SO4. Column chromato-
graphy was carried out on Silica Gel 60 (E. Merck). 2-
(Trimethylsilyl)ethyl b-D-galactopyranoside (5), 2,3,4,6-
tetra-O-benzoyl-a-D-galactopyranosyl trichloroacetimi-
date (9), phenyl 2,3,4-tri-O-benzoyl-1-thio-b-D-galacto-
pyranoside (10) and phenyl 2,3,4-tri-O-benzyl-1-thio-b-
L-fucopyranoside (18) were prepared by literature meth-
ods.23,25,26,28 Benzoylceramide 12 was prepared from N-
palmitoyl-D-erythro-sphingosine, which was purchased
from Acros Organics (Belgium) by the conventional
four-step procedure.31
3.3. 2-(Trimethylsilyl)ethyl 2,4-di-O-benzoyl-3-O-chloro-
acetyl-6-O-levulinoyl-b-D-galactopyranoside (7)
A solution of 6 (1.41g, 2.08mmol) in 2:1 THF–MeOH
(15mL) was hydrogenolyzed in the presence of 10%
Pd–C (500mg) for 4h at rt, then filtered and concen-
trated. The product was purified by silica gel column

2754
T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
chromatography using 5:1 toluene–acetone as eluent to
give 2-(trimethylsilyl)ethyl 2,4-di-O-benzoyl-6-O-levuli-
noyl-b-D-galactopyranoside (1.20g, quant). To a solu-
tion of this compound (349mg, 0.595mmol) in CH2Cl2
(1mL) was added ClCH2COCl (0.10mL, 1.26mmol)
and pyridine (0.10mL). The reaction mixture was stirred
for 2h at rt, then extracted with CHCl3, washed with 5%
HCl, aq NaHCO3 and water, dried (MgSO4) and con-
centrated. The product was purified by silica gel column
69.3 (C-5), 68.0 (C-4), 67.5 (C-3), 61.6 (C-6),
40.4, 37.8, 29.8, 27.7. HRFABMS: Calcd for
C29H27Cl4NO11Na [M+Na]+: m/z 728.0236. Found:
m/z 728.0266.
3.5. Phenyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyranos-
yl-(1!6)-2,3,4-tri-O-benzoyl-1-thio-b-D-galactopyrano-
side (11)
chromatography using 30:1 toluene–acetone as eluent to
To a solution of 9 (190mg, 0.26mmol) and 10 (100mg,
0.17mmol) in dry CH2Cl2 (3mL) was added powdered
4A MS (300mg), and the mixture was stirred for 2h at
rt, then cooled to 0ꢁC. TMSOTf (46lL, 0.25mmol)
was added to the mixture, which was stirred for 1h at
0ꢁC, then neutralized with Et3N. The solids were filtered
off and washed with CHCl3. The combined filtrate and
washings were successively washed with water, dried
(MgSO4) and concentrated. The product was purified
24
give compound 7 (354mg, 89.7%). ½aꢀ +37.6 (c 1.0,
D
CHCl3); 1H NMR (CDCl3): d 8.19–7.33 (m, 10H,
Ph·2), 5.77 (d, 1H, J3,4 3.1Hz, H-4), 5.63 (dd, 1H,
J1,2 7.9Hz, H-2), 5.45 (dd, 1H, J2,3 10.4Hz, H-3), 4.80
(d, 1H, H-1), 4.44 (dd, 1H, J5,6a 6.7Hz, J6a,6b 11.6Hz,
H-6a), 4.24 (dd, 1H, J5,6b 6.7Hz, H-6b), 4.17 (t, 1H,
H-5), 4.13–4.07 (m, 1H, –OCH2CH2–), 3.97–3.88 (each
d, 2H, Jgem 15.3Hz, ClCH2CO–), 3.72–3,67 (m, 1H,
–OCH2CH2–), 2.79 (t, 2H, –OCOCH2CH2COCH3),
2.61 (t, 2H, –OCOCH2CH2COCH3), 2.24 (s, 3H,
by silica gel column chromatography using 70:1 tolu-
ene–acetone as eluent to give 11 (181mg, 91.0%). ½aꢀ
24
D
1
–OCOCH2CH2COCH3),
1.03–0.92
(m,
2H,
+235.3 (c 0.3, 1 CHCl3); H NMR (CDCl3): d 8.11–
–OCH2CH2–), ꢁ0.09 (s, 9H, –Si(CH3)3); 13C NMR
(CDCl3): d 206.3, 172.1, 166.8, 165.9, 165.0, 133.7,
133.3, 130.0, 129.7, 129.3, 128.7, 128.6, 128.4, 100.9,
73.0, 70.7, 69.4, 67.8, 67.6, 61.5, 40.4, 37.8, 29.7, 27.8,
18.0. MALDI-TOFMS: Calcd for C32H39ClO11Si: m/z
662. Found: m/z 685 [M+Na]+. HRFABMS: Calcd for
C32H39ClO11SiNa [M+Na]+: m/z 685.1848. Found: m/z
685.1855.
7.20 (m, 40H, Ph·8), 5.95 (d, 1H, J3 ,4 3.7Hz, H-40),
0
0
0
0
5.91 (d, 1H, J3,4 3.7Hz, H-4), 5.79 (dd, 1H, J1 ,2
7.9Hz, J2 ,3 10.4Hz, H-20), 5.68 (t, 1H, J1,2 10.4Hz,
J2,3 10.4Hz, H-2), 5.57 (dd, 1H, H-30), 5.51 (dd, 1H,
H-3), 4.91 (d, 1H, H-10), 4.89 (d, 1H, H-1), 4.41 (dd,
0
0
1H, J5 ,6 b 6.1Hz, J6 a,6 b 11.0Hz, H-60a), 4.27–4.21 (m,
3H, H-5, H-50, H-60b), 4.09 (dd, 1H, J5,6a 4.9Hz, J6a,6b
11.0Hz, H-6a), 3.96 (dd, 1H, J5,6b 4.9Hz, H-6b); 13C
NMR (CDCl3): d 165.9, 165.5, 165.4, 165.2, 165.1,
134.1, 133.6, 133.5, 133.5, 133.4, 133.2, 133.1, 130.9,
130.1, 130.1, 129.8, 129.8, 129.3, 129.0, 129.0, 128.9,
128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 101.2
(C-10), 85.3 (C-1), 76.8 (C-5), 73.0 (C-3), 71.7 (C-30),
71.3 (C-50), 69.7 (C-20), 68.5 (C-4), 67.9 (C-6), 67.8 (C-
40), 67.7 (C-2), 61.7 (C-60). MALDI-TOFMS:
Calcd for C67H54O17S: m/z 1162. Found: m/z 1185
[M+Na]+.
0
0
0
0
3.4. 2,4-Di-O-benzoyl-3-O-chloroacetyl-6-O-levulinoyl-a-
D-galactopyranosyl trichloroacetimidate (8)
To a solution of 7 (84mg, 0.13mmol) in CH2Cl2 (1mL)
cooled to 0ꢁC was added CF3COOH (1mL), and the
mixture was stirred for 2h at rt and concentrated.
EtOAc and toluene (1:2) were added and then evapo-
rated to give the 1-hydroxy compound. To a solution
of the residue in CH2Cl2 (2mL) cooled at 0ꢁC were
added trichloroacetonitrile (0.27mL, 2.69mmol) and
DBU (30.0lL, 0.20mmol). The mixture was stirred for
1h at 0ꢁC. After completion of the reaction, the mixture
was concentrated. Column chromatography (30:1 tolu-
3.6. 2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl-
(1!6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-(1!1)-
(2S,3R)-3-O-benzoyl-2-hexadecanamido-octadecane-1,3-
diol (13)
ene–acetone) of the residue on silica gel gave 8 (66mg,
24
D
73.7%) as an amorphous mass. ½aꢀ +122.0 (c 0.5,
To a solution of 11 (50mg, 43.0lmol) and 12 (33mg,
51.2lmol) in dry CH2Cl2 (1.5mL) was added powdered
4A MS (150mg), and the mixture was stirred for 2h at
rt, then cooled to ꢁ40ꢁC. NIS (15mg, 66.7lmol) and
TfOH (1.5lL, 17.0lmol) were added to the mixture,
which was stirred for 2h at ꢁ40ꢁC, then neutralized
with Et3N. The solids were filtered off and washed with
CHCl3. The combined filtrate and washings were succes-
sively washed with aq Na2S2O3 and water, dried
(MgSO4) and concentrated. The product was purified
1
CHCl3); H NMR (CDCl3): d 8.65 (s, 1H, NH), 8.11–
7.26 (m, 10H, Ph·2), 6.84 (d, 1H, J1,2 7.9Hz, H-1),
5.87 (d, 1H, J3,4 3.1Hz, H-4), 5.84 (dd, 1H, J2,3
11.0Hz, H-3), 5.74 (dd, 1H, H-2), 4.64 (t, 1H, J5,6b
J5,6a 6.1Hz, H-5), 4.29 (dd, 1H, J6a,6b 11.0Hz, H-6a),
4.24 (dd, 1H, H-6b), 3.96–3.88 (each d, 2H, Jgem
15.3Hz, ClCH2CO–), 2.69 (t, 2H, –OCOCH2CH2-
COCH3), 2.53 (t, 2H, –OCOCH2CH2COCH3), 2.15 (s,
3H, –OCOCH2CH2COCH3); 13C NMR (CDCl3): d
206.2, 172.1, 166.7, 165.8, 165.5, 160.6, 134.0, 133.8,
130.0, 129.9, 128.8, 128.7, 128.6, 93.5 (C-1), 69.6 (C-2),
by silica gel column chromatography using 20:1 tolu-
ene–EtOAc as eluent to give 13 (32mg, 43.9%). ½aꢀ
24
D

T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
2755
1
+69.2 (c 0.8, CHCl3); H NMR (CDCl3) d: 4.55 (d, 1H,
DI-TOFMS: Calcd for C60H53ClO18S: m/z 1128. Found:
m/z 1151 [M+Na]+. HRFABMS: Calcd for
C60H54ClO18S [M+H]+: m/z 1129.2719. Found: m/z
1129.2684.
J1 ,2 7.3Hz, H-10), 4.41(d, 1H, J1,2 7.9Hz, H-1). MAL-
DI-TOFMS: Calcd for C102H119NO21: m/z 1693. Found:
m/z 1716 [M+Na]+.
0
0
3.7. b-D-Galactopyranosyl-(1!6)-b-D-galactopyranosyl-
3.9. Phenyl 2,4-di-O-benzoyl-6-O-levulinoyl-b-D-galacto-
pyranosyl-(1!6)-2,3,4-tri-O-benzoyl-1-thio-b-D-galacto-
pyranoside (15)
(1!1)-(2S,3R)-2-hexadecanamido-octadecane-1,3-diol
(1)
To a solution of 12 (32mg, 18.9lmol) in 1:1 MeOH–1,4-
dioxane (2mL) was added NaOMe (40mg), and the
mixture was stirred overnight 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
To a solution of 14 (50mg, 44.2lmol) in 6:1 pyridine–
EtOH (3.5mL) was added thiourea (34mg, 0.45mmol),
and the mixture was stirred for 1h at rt. The mixture
was diluted with CHCl3, washed with NaHCO3 and
water, dried (MgSO4) and concentrated. The product
was purified by silica gel column chromatography using
7:1 toluene–acetone as eluent to give 15 (35mg, 75.1%).
chromatography (1:1 CHCl3–MeOH) of the residue on
24
D
24
1
Sephadex LH-20 gave 1 (16mg, quant). ½aꢀ +6.8 (c
½aꢀ +132.0 (c 0.3, CHCl3); H NMR (CDCl3): d 8.13–
D
0.6, 1:1 CHCl3–MeOH); 1H NMR (49:1 DMSO-d6–
7.16 (m, 30H, Ph·6), 5.82 (d, 1H, J3,4 3.1Hz, H-4),
5.64 (t, 1H, J1,2 9.8Hz, J2,3 10.4Hz, H-2), 5.57 (d, 1H,
D2O) d: 4.15 (d, 1H, J1 ,2 7.3Hz, H-10), 4.07 (d, 1H,
J1,2 7.3Hz, H-1). MALDI-TOFMS: Calcd for
C46H89NO13: m/z 863. Found: m/z 886 [M+Na]+. HRF-
ABMS: Calcd for C46H89NO13Na [M+Na]+: m/z
886.6232. Found: m/z 886.6153.
0
0
J3 ,4 3.7Hz, H-40), 5.45 (dd, 1H, H-3), 5.29 (d, 1H,
0
0
J1 ,2 7.9Hz, J2 ,3 10.4Hz, H-20), 4.92 (d, 1H, H-1),
4.76 (d, 1H, H-10), 4.31 (dd, 1H, H-5), 4.12–3.90 (m,
6H, H-3, H-50, H-6a, H-6b, H-6a0, H-6b0), 2.70 (t, 2H,
0
0
0
0
–OCOCH2CH2COCH3),
2.49–2.41
(m,
2H,
3.8. Phenyl 2,4-di-O-benzoyl-3-O-chloroacetyl-6-O-levu-
linoyl-b-D-galactopyranosyl-(1!6)-2,3,4-tri-O-benzoyl-1-
thio-b-D-galactopyranoside (14)
–OCOCH2CH2COCH3), 2.13 (d, 1H, OH), 2.10 (s,
3H, –OCOCH2CH2COCH3); 13C NMR (CDCl3): d
172.4, 167.0, 166.2, 165.4, 165.2, 133.6, 133.4, 133.3,
133.1, 131.4, 130.1, 130.0, 129.9, 129.8, 129.7, 129.4,
129.0, 128.8, 128.6, 128.5, 128.4, 128.4, 128.20, 101.3
(C-10), 85.4 (C-1), 76.7 (C-5), 73.7 (C-50), 73.1 (C-3),
72.0 (C-30), 71.3 (C-6), 70.4 (C-40), 68.7 (C-20,C-4),
67.9 (C-2), 63.2 (C-60), 38.0, 29.7, 27.9. MALDI-
TOFMS: Calcd for C58H52O17S: m/z 1052. Found:
m/z 1075 [M+Na]+. HRFABMS: Calcd for
C58H53O17S [M+H]+: m/z 1053.3003. Found: m/z
1053.3073.
To a solution of 8 (140mg, 0.20mmol) and 10 (77mg,
0.13mmol) in dry CH2Cl2 (2mL) was added powdered
4A MS (300mg), and the mixture was stirred for 2h at
rt, then cooled to 0ꢁC. TMSOTf (7.2lL, 39.8lmol)
was added to the mixture, which was stirred for 30min
at 0ꢁC, then neutralized with Et3N. The solids were fil-
tered off and washed with CHCl3. The combined filtrate
and washings were successively washed with water, dried
(MgSO4) and concentrated. The product was purified by
silica gel column chromatography using 15:1 toluene–
acetone as eluent to give 14 (145mg, quant). ½aꢀ
3.10. Phenyl 2,4-di-O-benzoyl-3-O-chloroacetyl-b-D-
galactopyranosyl-(1!6)-2,3,4-tri-O-benzoyl-1-thio-b-D-
galactopyranoside (16)
24
D
1
+96.3 (c 0.3, CHCl3); H NMR (CDCl3): d 8.14–7.20
(m, 30H, Ph·6), 5.84 (d, 1H, J3,4 3.7Hz, H-4), 5.67–
5.64 (m, 2H, H-2, H-40), 5.58 (dd, 1H, J1 ,2 7.9Hz,
To a solution of 14 (50mg, 44.3lmol) in 2:1 MeOH–
THF (1.7mL) was added hydrazine acetate (4.08mg,
44.3lmol), and the mixture was stirred for 3h at rt.
The mixture was diluted with CHCl3, was washed with
aq NaHCO3 and water, dried (MgSO4) and concen-
trated. The product was purified by silica gel column
0
0
J2 ,3 10.4Hz, H-20), 5.46 (dd, 1H, J2,3 9.8Hz, H-3),
5.35 (dd, 1H, H-30), 4.90 (d, 1H, J1,2 9.8Hz, H-1), 4.82
0
0
(d, 1H, H-10), 4.27 (dd, 1H, J5 ,6a 4.9Hz, J6a ,6b
0
0
0
0
7.32Hz, H-6a0), 4.12–3.94 (m, 5H, H-5, H-50, H-6a,
H-6b, H-6b0), 3.91–3.83 (each d, 2H, Jgem 15.3Hz,
ClCH2CO–), 2.72 (t, 2H, –OCOCH2CH2COCH3),
2.49 (dd, 2H, –OCOCH2CH2COCH3), 2.17 (s, 3H,
–OCOCH2CH2COCH3); 13C NMR (CDCl3): d 147.3,
134.0, 133.8, 133.5, 133.3, 130.2, 129.9, 129.8, 129.8,
129.4, 129.1, 128.9, 128.8, 128.5, 128.4, 128.2, 113.0,
109.3, 101.4 (C-10), 91.8, 85.4 (C-1), 76.8 (C-5), 73.1
(C-3), 72.9 (C-30), 71.0 (C-50), 69.3 (C-20), 68.7 (C-4),
68.5 (C-6), 67.9 (C-40), 67.6 (C-2), 61.5 (C-60), 52.2,
48.3, 40.4, 38.0, 30.9, 29.7, 27.9, 22.7, 22.0, 11.9. MAL-
chromatography using 20:1 toluene–acetone as eluent
24
to give 16 (40mg, 87.6%). ½aꢀ +97.4 (c 1.1, CHCl3);
D
1H NMR (CDCl3): d 8.07–7.12 (m, 30H, Ph·6), 5.87
(d, 1H, J3,4 3.1Hz, H-4), 5.59–5.51 (m, 3H, H-2, H-20,
H-40), 5.41 (dd, 1H, J2,3 9.8Hz, H-3), 5.30 (dd, 1H,
J2,3 10.4Hz, J3,4 3.1Hz, H-30), 4.79 (d, 1H, J1,2 9.8Hz,
H-1), 4.70 (d, 1H, J1 ,2 7.3Hz, H-10), 4.10 (t, 1H, J5,6b
6.1Hz, J5,6a 5.5Hz, H-5), 3.91 (dd, 1H, J6a,6b 11.0Hz,
H-6a) 3.87–3.74 (m, 4H, H-50, H-6b, ClCH2CO–), 3.55
0
0

2756
T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
(dd, 1H, J5 ,6a 6.7Hz, J6a ,6b 11.6Hz, H-6a0), 3.38 (dd,
which was stirred for 2h at ꢁ65ꢁC, then neutralized
with Et3N. The solids were filtered off and washed with
CHCl3. The combined filtrate and washings were succes-
sively washed with aq Na2S2O3 and water, dried
(MgSO4) and concentrated. The product was purified
0
0
0
0
1H, J5 ,6b 5.5Hz, H-6b0), 2.09 (br s, 1H, OH); 13C
NMR (CDCl3): d 166.8, 166.6, 165.5, 165.3, 165.2,
165.1, 134.4, 133.9, 133.5, 133.5, 133.3, 133.2, 130.6,
130.2, 129.9, 129.7, 129.3, 129.1, 128.9, 128.8, 128.8,
128.7, 128.5, 128.4, 128.2, 101.3 (C-10), 85.3 (C-1), 76.8
(C-5), 73.9 (C-50), 73.1 (C-3), 72.9 (C-30), 69.6 (C-20),
68.5 (C-4), 68.2 (C-40), 67.9 (C-2), 67.8 (C-6), 60.7 (C-
60), 40.4, 29.7. MALDI-TOFMS: Calcd for
C55H47ClO16S: m/z 1030. Found: m/z 1053 [M+Na]+.
HRFABMS: Calcd for C55H48ClO16S [M+H]+: m/z
1031.2352. Found: m/z 1031.2356.
0
0
by silica gel column chromatography using 4:3 hex-
ane–EtOAc as eluent to give 19 (100mg, 78.5%). ½aꢀ
24
D
1
+68.1 (c 1.4, CHCl3); H NMR (CDCl3): d 8.15–6.94
(m, 45H, Ph·9), 5.83 (d, 1H, H-4), 5.70–5.60 (m, 3H,
H-20, H-2, H-40), 5.45 (dd, 1H, J2,3 9.8Hz, H-3), 5.05
(d, 1H, J1 ,2 2.0Hz, H-100), 4.89 (d, 1H, J1,2 9.8Hz, H-
1), 4.81 and 4.49 (each d, 2H, Jgem 11.6Hz, benzylic
00 00
methylene), 4.76 (d, 1H, J1 ,2 7.9Hz, H-10), 4.38–3.85
(m, 12H, benzylic methylene·2, H-6a, H-6b, H-6a0,
0
0
3.11. Phenyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyranos-
yl-(1!6)-2,4-di-O-benzoyl-3-O-chloroacetyl-b-D-galacto-
pyranosyl-(1!6)- 2,3,4-tri-O-benzoyl-1-thio-b-D-
galactopyranoside (17)
H-6b0, H-30, H-5, H-50, H-500), 3.78 (dd, 1H, J2 ,3
00 00
10.4Hz, H-200), 3.63 (dd, 1H, H-300), 3.49 (s, 1H, H-400),
2.74 (t, 2H, –OCOCH2CH2COCH3), 2.52 (t, 2H,
–OCOCH2CH2COCH3), 2.13 (s, 3H, –OCOCH2CH2-
COCH3), 1.01 (d, 3H, H-600); 13C NMR (CDCl3): d
206.8, 172.6, 166.3, 165.4, 165.3, 165.1, 164.8, 139.0,
138.6, 138.5, 133.8, 133.4, 133.4, 133.3, 133.1, 133.1,
130.2, 129.9, 129.9, 129.8, 129.7, 129.5, 129.4, 129.1,
128.9, 128.6, 128.5, 128.4, 128.4, 128.4, 128.2,
128.2, 128.1, 128.0, 127.5, 127.3, 127.2, 127.1,
101.9 (C-10), 98.8 (C-100), 85.4 (C-1), 78.9 (C-300),
78.0 (C-400), 76.8 (C-5) 75.2 (C-200), 75.1 (C-30), 74.7
(benzylic methylene), 73.3 (benzylic methylene), 73.1
(C-3), 72.0 (benzylic methylene), 71.9, 71.6, 69.9, 68.9
(C-4), 68.7 (C-6), 68.0 (C-20), 67.7 (C-2), 62.5
(C-60), 38.1, 29.7, 28.0, 16.4. MALDI-TOFMS:
Calcd for C85H80O21S: m/z 1468. Found: m/z 1491
[M+Na]+.
To a solution of 16 (150mg, 0.14mmol) and 9 (162mg,
0.22lmol) in dry CH2Cl2 (3mL) was added powdered
4A MS (300mg), and the mixture was stirred for 2h at
rt, then cooled to 0ꢁC. TMSOTf (7.8lL, 43.1lmol)
was added to the mixture, which was stirred for 1h at
0ꢁC, then neutralized with Et3N. The solids were filtered
off and washed with CHCl3. The combined filtrate and
washings were successively washed with water, dried
(MgSO4) and concentrated. The product was purified
by silica gel column chromatography using 80:1 tolu-
24
ene–acetone as eluent to give 17 (215mg, 91.8%). ½aꢀ
D
1
+93.3 (c 0.3, CHCl3); H NMR (CDCl3): d 8.12–7.20
(m, 50H, Ph·10), 5.89 (d, 1H, J3,4 3.1Hz, H-4), 5.87
(d, 1H, J3 ,4 3.1Hz, H-40), 5.78 (d, 1H, J3 ,4 3.1Hz,
H-400), 5.67–5.32 (m, 6H, H-2, H-20, H-200, H-3, H-30,
0
0
00 00
H-300), 4.88 (d, 1H, J1,2 9.8Hz, H-1), 4.69 (d, 1H, J1 ,2
3.13. 2,3,4-Tri-O-benzyl-a-L-fucopyranosyl-(1!3)-2,4-
di-O-benzoyl-6-O-levulinoyl-b-D-galactopyranosyl-
(1!6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-
(2S,3R)-3-O-benzoyl-2-hexadecan-amido-octadecane-1,3-
diol (20)
0
0
7.9Hz, H-10), 4.63 (d, 1H, J1 ,2 7.3Hz, H-100), 4.15–
3.54 (m, 11H, H-5, H-50, H-500, H-6a, H-6b, H-60a, H-
60b, H-600a, H-600b, ClCH2CO–); 13C NMR (CDCl3): d
166.6, 165.7, 165.5, 165.4, 165.3, 165.1, 165.0, 163.4,
134.0, 133.6, 133.3, 133.2, 130.9, 130.1, 130.0, 129.9,
129.8, 129.7, 129.7, 129.3, 129.2, 129.1, 129.0, 128.9,
128.8, 128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.2,
100.9, 100.5, 76.7, 73.0, 72.8, 71.9, 71.5, 71.0, 69.8,
69.4, 68.4, 67.8, 67.7, 67.2, 65.6, 61.2, 40.5. MALDI-
TOFMS: Calcd for C89H73ClO25S: m/z 1608. Found:
m/z 1631 [M+Na]+.
00 00
To a solution of 19 (96mg, 65lmol) and 12 (87mg,
0.14lmol) in dry CH2Cl2 (2mL) was added powdered
AW300 MS (200mg), and the mixture was stirred for
2h at rt, then cooled to 0ꢁC. NIS (23mg, 102lmol)
and TfOH (3lL, 34lmol) were added to the mixture,
which was stirred for 2h at 0ꢁC, then neutralized with
Et3N. The solids were filtered off and washed with
CHCl3. The combined filtrate and washings were succes-
sively washed with aq Na2S2O3 and water, dried
(MgSO4) and concentrated. The product was purified
3.12. Phenyl 2,3,4-tri-O-benzyl-a-L-fucopyranosyl-
(1!3)-2,4-di-O-benzoyl-6-O-levulinoyl-b-D-galactopy-
ranosyl-(1!6)-2,3,4-tri-O-benzoyl-1-thio-b-D-galactopyr-
anoside (19)
by silica gel column chromatography using 3:2 hex-
ane–EtOAc as eluent to give 20 (70mg, 53.5%). ½aꢀ
24
D
To a solution of 15 (100mg, 86.7lmol) and 18 (100mg,
190lmol) in dry CH2Cl2 (2mL) was added powdered
4A MS (200mg), and the mixture was stirred for 2h at
rt, then cooled to ꢁ65ꢁC. NIS (70mg, 0.31lmol) and
TfOH (1.7lL, 19lmol) were added to the mixture,
+38.9 (c 0.9, CHCl3); 1H NMR (CDCl3): d 5.08 (d,
1H, J1 ,2 3.7Hz, H-100), 4.55 (d, 1H, J1 ,2 7.9Hz, H-
10), 4.20 (d, 1H, J1,2 7.9Hz, H-1). MALDI-TOFMS:
Calcd for C120H147NO25: m/z 2002. Found: m/z 2025
[M+Na]+.
00 00
0
0

T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
2757
3.14. a-L-Fucopyranosyl-(1!3)-2,4-di-O-benzoyl-6-O-
7.9Hz, H-1). MALDI-TOFMS: Calcd for C124H140
ClNO29: m/z 2141. Found: m/z 2164 [M+Na]+.
-
levulinoyl-b-D-galactopyranosyl-(1!6)-2,3,4-tri-O-benz-
oyl-b-D-galactopyranosyl-(1!1)-(2S,3R)-3-O-benzoyl-
2-hexadecanamido-octadecane-1,3-diol (21)
3.17. b-D-Galactopyranosyl-(1!6)-b-D-galactopyranosyl-
(1!6)-b-D-galactopyranosyl-(1!1)-(2S,3R)-2-hexadecan-
amido-octadecane-1,3-diol (3)
A solution of 20 (70mg, 35lmol) in 2:1 THF–MeOH
(4.5mL) was hydrogenolyzed in the presence of 10%
Pd–C (120mg) overnight at rt, then filtered and concen-
trated. The product was purified by silica gel column
chromatography using 60:1 CHCl3–MeOH as eluent to
To a solution of 22 (35mg, 16.3lmol) in 1:1 MeOH–1,4-
dioxane (1mL) was added NaOMe (40mg), and the
mixture was stirred overnight 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
24
D
1
give 21 (51mg, 84.2%). ½aꢀ +91.1 (c 0.7, CHCl3); H
NMR (CDCl3): d 4.93 (d, 1H, J1 ,2 3.7Hz, H-100), 4.64
00 00
(d, 1H, J1 ,2 7.3Hz, H-10), 4.45 (d, 1H, J1,2 6.7Hz, H-
1). MALDI-TOFMS: Calcd for C99H129NO25: m/z
1731. Found: m/z 1754 [M+Na]+.
0
0
chromatography (1:1 CHCl3–MeOH) of the residue on
24
D
Sephadex LH-20 gave 3 (17mg, quant). ½aꢀ +9.2 (c
1
0.4, CHCl3); H NMR (49:1 DMSO-d6–D2O): d 4.17
(d, 1H, J1 ,2 7.3Hz, H-10), 4.15 (d, 1H, J1 ,2 6.7Hz,
H-100), 4.07 (d, 1H, J1,2 7.3Hz, H-1). MALDI-TOFMS:
Calcd for C52H99NO18: m/z 1025. Found: m/z 1048
[M+Na]+. HRFABMS: Calcd for C52H99NO18Na
[M+Na]+: m/z 1048.6760. Found m/z 1048.6816.
0
0
00 00
3.15. a-L-Fucopyranosyl-(1!3)-b-D-galactopyranosyl-
(1!6)-b-D-galactopyranosyl-(1!1)-(2S,3R)-2-hexadecan-
amido-octadecane-1,3-diol (2)
To a solution of 21 (26mg, 15lmol) in 1:1 MeOH–1,4-
dioxane (2mL) was added NaOMe (20mg), and the
mixture was stirred overnight 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
3.18. Phenyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyranos-
yl-(1!6)-2,4-di-O-benzoyl-b-D-galactopyranosyl-(1!6)-
2,3,4-tri-O-benzoyl-1-thio-b-D-galactopyranoside (23)
chromatography (1:1 CHCl3–MeOH) of the residue on
To a solution of 17 (256mg, 159lmol) in 6:1 pyridine–
EtOH (3mL) was added thiourea (60mg, 788lmol),
and the mixture was stirred for 1h at 60ꢁC. The mixture
was was diluted with CHCl3, washed with NaHCO3 and
water, dried (MgSO4) and concentrated. The product
was purified by silica gel column chromatography using
20:1 toluene–acetone as eluent to give 23 (204mg,
24
D
Sephadex LH-20 gave 2 (11mg, 72.6%). ½aꢀ +7.0 (c
0.3, 1:1 CHCl3–MeOH); 1H NMR (49:1 DMSO-d6–
D2O): d 4.91 (d, 1H, J1 ,2 3.7Hz, H-100), 4.24 (d, 1H,
00 00
J1 ,2 7.9Hz, H-10), 4.07 (d, 1H, J1,2 7.3Hz, H-1). MAL-
DI-TOFMS: Calcd for C52H99NO17: m/z 1009. Found:
m/z 1032 [M+Na]+. HRFABMS: Calcd for
C52H99NO17Na [M+Na]+: m/z 1032.6811. Found: m/z
1032.6766.
0
0
24
1
83.7%). ½aꢀ +86.5 (c 0.5, CHCl3); H NMR (CDCl3):
D
d 8.13–7.14 (m, 50H, Ph·10), 5.89 (d, 2H, J3,4 3.1Hz,
J3 ,4 3.1Hz, H-4, H-400), 5.72–5.50 (m, 5H, H-2, H-200,
00 00
H-3, H-300, H-40), 5.26 (dd, 1H, J1 ,2 9.8Hz, J2 ,3
0
0
0
0
3.16. 2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl-
(1!6)-2,4-di-O-benzoyl-3-O-chloroacetyl-b-D-galactopyr-
anosyl-(1!6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-
(1!1)-(2S,3R)-3-O-benzoyl-2-hexadecanamido-octadec-
ane-1,3-diol (22)
7.9Hz, H-20), 4.91 (d, 1H, J1,2 9.8Hz, H-1), 4.65 (d,
1H, J1 ,2 9.8Hz, H-100), 4.63 (d, 1H, J1 ,2 7.9Hz, H-
10), 4.24–3.51 (m, 10H, H-30, H-5, H-50, H-500, H-6a,
H-6b, H-60a, H-60b, H-600a, H-600b); 13C NMR (CDCl3):
d 166.6, 166.3, 165.8, 165.5, 165.3, 165.1, 165.0, 133.8,
133.6, 133.4, 133.3, 133.1, 131.2, 130.1, 129.9, 129.8,
129.7, 129.5, 129.3, 129.3, 129.0, 128.9, 128.79, 128.7,
128.6, 128.4, 128.4, 128.2, 128.2, 101.0, 100.8, 85.5,
76.7, 73.4, 73.0, 72.4, 71.8, 71.5, 71.1, 70.2, 69.8, 68.5,
67.9, 67.5, 66.8, 61.5. MALDI-TOFMS: Calcd for
C87H72O24S: m/z 1532. Found: m/z 1555 [M+Na]+.
00 00
0
0
To a solution of 17 (100mg, 62.1lmol) and 12 (56mg,
87lmol) in dry CH2Cl2 (1.5mL) was added powdered
4A MS (200mg), and the mixture was stirred for 2h at
rt, then cooled to 0ꢁC. NIS (28mg, 0.12mmol) and
TfOH (2.8lL, 32lmol) were added to the mixture, which
was stirred for 2h at 0ꢁC, 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 (MgSO4)
and concentrated. The product was purified by silica
3.19. Phenyl 2,3,4,6-tetra-O-benzoyl-b-D-galactopyranos-
yl-(1!6)-[2,3,4-tri-O-benzyl-a-L-fuco-pyranosyl-
(1!3)]-2,4-di-O-benzoyl-b-D-galactopyranosyl-(1!6)-
2,3,4-tri-O-benzoyl-1-thio-b-D-galactopyranoside (24)
gel column chromatography using 20:1 toluene–EtOAc
24
D
as eluent to give 22 (44mg, 33.0%). ½aꢀ +37.6 (c 0.9,
1
0
0
CHCl3); H NMR (CDCl3): d 4.62 (d, 1H, J1 ,2 7.9Hz,
To a solution of 23 (63mg, 41.1lmol) and 18 (43mg,
81.6lmol) in dry CH2Cl2 (1mL) was added powdered
H-10), 4.54 (d, 1H, J1 ,2 7.9Hz, H-100), 4.18 (d, 1H, J1,2
00 00

2758
T. Yamamura et al. / Carbohydrate Research 339 (2004) 2749–2759
4A MS (100mg), and the mixture was stirred for 2h at
rt, then cooled to ꢁ60ꢁC. NIS (28mg, 124lmol) and
TfOH (0.7lL, 7.91lmol) were added to the mixture,
which was stirred for 1h at ꢁ60ꢁC, then neutralized
with Et3N. The solids were filtered off and washed with
CHCl3. The combined filtrate and washings were succes-
sively washed with aq Na2S2O3 and water, dried
(MgSO4) and concentrated. The product was purified
3.22. b-D-Galactopyranosyl-(1!6)-[a-L-fucopyranosyl-
(1!3)]-b-D-galactopyranosyl-(1!6)-b-D-galactopyranos-
yl-(1!1)-(2S,3R)-2-hexadecanamido-octadecane-1,3-
diol (4)
To a solution of 26 (20mg, 9.04lmol) in 1:1 MeOH–1,4-
dioxane (1mL) was added NaOMe (20mg), and the
mixture was stirred for 14h 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
by silica gel column chromatography using 10:1 tolu-
24
ene–EtOAc as eluent to give 24 (73mg, 91.1%). ½aꢀ
D
+36.1 (c 0.9, CHCl3); 1H NMR (CDCl3): d 5.06 (d,
1H, J1,2 3.7Hz, Fuc of H-1), 4.83 (d, 1H, J1,2 9.2Hz,
chromatography (1:1 CHCl3–MeOH) of the residue on
24
D
H-1), 4.68 (d, 1H, J1 ,2 7.9Hz, H-10), 4.57 (d, 1H,
Sephadex LH-20 gave 4 (10.0mg, 94.4%). ½aꢀ ꢁ11.0
0
0
13
1
(c 0.3, 1:1 CHCl3–MeOH); H NMR (49:1 DMSO-d6–
J1 ,2 7.9Hz, H-100); C NMR (CDCl3): d 101.3 (C-10),
00 00
101.1 (C-100), 98.5 (Fuc of C-1), 85.4 (C-1). MALDI-
TOFMS: Calcd for C114H100O28S: m/z 1948. Found:
m/z 1971 [M+Na]+.
D2O): d 4.92 (d, 1H, J1,2 4.3Hz, Fuc of H-1), 4.25 (d,
1H, J1 ,2 7.3Hz, H-100), 4.16 (d, 1H, J1 ,2 7.3Hz, H-
10), 4.07 (d, 1H, J1,2 7.3Hz, H-1). MALDI-TOFMS:
Calcd for C58H109NO22: m/z 1171. Found: m/z 1194
[M+Na]+. HRFABMS: Calcd for C58H109NO22Na
[M+Na]+: m/z 1194.7339. Found: m/z 1194.7284.
00 00
0
0
3.20. 2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl-
(1!6)-[2,3,4-tri-O-benzyl-a-L-fucopyranosyl-(1!3)]-2,4-
di-O-benzoyl-b-D-galactopyranosyl-(1!6)-2,3,4-tri-O-
benzoyl-b-D-galactopyranosyl-(1!1)-(2S,3R)-3-O-benz-
oyl-2-hexadecanamido-octadecane-1,3-diol (25)
Acknowledgements
This work was supported by a Grant-in-Aid for
Scientific Research (No. 14771250) from the Ministry
of Education, Science, Sports and Culture of Japan.
The authors are grateful to Ms. J. Hada for providing
NMR and MS data.
To a solution of 24 (68mg, 34.9lmol) and 12 (45mg,
69.9lmol) in dry CH2Cl2 (1.5mL) was added powdered
4A MS (150mg), and the mixture was stirred for 2h at
rt, then cooled to ꢁ40ꢁC. NIS (16mg, 71.1lmol) and
TfOH (1.6lL, 18.1lmol) were added to the mixture,
which was stirred for 2h at ꢁ40ꢁC, then neutralized
with Et3N. The solids were filtered off and washed with
CHCl3. The combined filtrate and washings were succes-
sively washed with aq Na2S2O3 and water, dried
(MgSO4) and concentrated. The product was purified
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