Synthetic studies on glycosphingolipids from the Protostomia phyla: Syntheses of arthro-series glycosphingolipids

Article abstract of DOI:10.1016/S0008-6215(02)00295-1

Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing GlcNAcβ-(1→3)-Man and GalNAcβ-(1→4)-GlcNAcβ-(1→3)-Man. We have synthesized two glycosphingolipids, β-D-GlcNAcp-(1→3)-β-D-Manp-(1→4)-β-

Full text of DOI:10.1016/S0008-6215(02)00295-1

Carbohydrate Research 337 (2002) 2037–2047
www.elsevier.com/locate/carres
Synthetic studies on glycosphingolipids from the Protostomia
phyla: syntheses of arthro-series glycosphingolipids
Isao Ohtsuka,a Noriyasu Hada,a Mutsumi Sugita,b Tadahiro Takedaa,*
aKyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
bDepartment of Chemistry, Faculty of Liberal Arts and Education, Shiga Uni6ersity, 2-5-1, Hiratsu, Otsu-shi, Shiga-ken 520-0862, Japan
Received 8 April 2002; accepted 28 May 2002
Dedicated to Professor Derek Horton on the occasion of his 70th birthday
Abstract
Glycosphingolipids isolated from larvae of the green-bottle fly, Lucilia caesar, have quite unique structures containing
GlcNAcb-(1“3)-Man and GalNAcb-(1“4)-GlcNAcb-(1“3)-Man. We have synthesized two glycosphingolipids, b- -GlcNAcp-
(1“3)-b- -Manp-(1“4)-b- -Glcp-(1“1)-Cer and b- -GalNAcp-(1“4)-b- -GlcNAcp-(1“3)-b- -Manp-(1“4)-b- -Glcp-
D
D
D
D
D
D
D
(1“1)-Cer. A key reaction in the synthetic sequence is the application of the intramolecular aglycon delivery (IAD) approach for
the synthesis of the b-mannopyranosidic linkages. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Glycosphingolipids; Arthro-series; Chemical synthesis; Lucilia caesar
1. Introduction
fertilization.1 They are divided into separate series
based on the nature of the core saccharide, such as
Globo-, Isoglobo-, Lacto-, Neolacto-, and Ganglio-se-
ries.2 Their core may be repeated or extended and
finally terminated with blood group and other antigenic
determinants, including sialic acid and sulfate groups.
On the other hand, the core saccharides of glycosph-
ingolipids from invertebrate animal species differ
greatly from those of mammals (Table 1). A unique
series of glycolipids having mannose, internally located
fucose, and O-methyl sugars have been found in vari-
ous invertebrates.3 These differences from common ver-
tebrate glycolipids have prompted the study of
glycolipid profiles of other unexplored invertebrates,
especially in the Protostomia phyla. Furthermore, the
biological function of these glycans has, for the most
part, not been explored. We have been interested in the
relationships between the structure and biological func-
tion of glycolipids from invertebrate animal species and
have so far synthesized oligosaccharides from various
P. phyla.4 Sugita et al. found some novel glycolipids
The structure and the biological role of the oligosac-
charide residues of glycosphingolipids in mammalian
tissues have been extensively studied and are known to
participate in various biological processes, i.e., im-
munological response, cell-growth regulation, a com-
mon metastatic inflammation, viral infection, and
Table 1
Glycosphingolipid series found in Protostomia phyla
Prefix of series Structure
Arthro
Mollu
b-D
b-D
a-D
b-D
a-D
b-D
b-D
-GlcpNAc-(1“3)-b-
D-Manp-(1“4)-
-Glcp
-Manp-(1“3)-b-
D-Manp-(1“4)-
-Glcp
Gara
Neogara
Schisto
-Galp-(1“6)-b-
D
-Galp
-Galp-(1“6)-b-D-Galp
-GalpNAc-(1“4)-b-D-Glcp
with
Glcp-(1“1)-Cer (1) and b-
GlcNAcp-(1“3)-b- -Manp-(1“4)-b-
Cer (2) in larvae of the green-bottle fly, Lucilia caesar,
b-
D
-GlcNAcp-(1“3)-b-
D
-Manp-(1“4)-b-
D
-
D
-GalNAcp-(1“4)-b-
D-
* Corresponding author. Tel.: +3-5400-2696; fax: +3-
5400-2556.
E-mail address: takeda-td@kyoritsu-ph.ac.jp (T. Takeda).
D
D
-Glcp-(1“1)-
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00295-1

2038
I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
Scheme 1. (a) p-MBnCl, Bu4NBr, CH2Cl2–NaOHaq; (b) TBDPSCl, imidazole, DMF; (c) LevOH, DCC, DMAP, CH2Cl2; (d) (1)
CF3COOH, CH2Cl2; (2) CCl3CN, DBU, CH2Cl2.
which belongs to the class Insecta5 (structures 1+2). It
is particularly interesting to note that these glycosphin-
golipids contain the unique structures GlcNAcb(1“
3)Man and GalNAcb(1“4)GlcNAcb(1“3)Man. The
structure of other glycolipids with longer sugar chains
has also been defined.6 These compounds have also
out as depicted in Scheme 1. Compound 5 was pre-
pared from known ethyl 4,6-O-benzylidene-1-thio-a-D-
mannopyranoside (3)10 by the following two-step
procedure. Regioselective p-methoxybenzylation at the
C-2 position of the starting material with p-methoxy-
benzyl chloride and tetrabutylammonium bromide, fol-
lowed by silylation with tert-butyldiphenylsilyl chloride
(TBDPS-Cl),9c gave compound 5. Imidate donor 8 was
obtained from 2-(trimethylsilyl)ethyl 3,6-di-O-benzyl-2-
1,2-cis-b-D-mannopyranosidic linkages, which are one
of the most difficult linkages to be synthesized7 and for
which many carbohydrate chemists8,9 have developed
methods to make these linkages. Furthermore, applica-
tion of these methods to the synthesis of asparagine
(Asn)-linked oligosaccharides and the glycolipids from
the fresh water bivalve Hyriopsis schlegelii is under
current investigation.9 Among those b-mannoglycoside
approaches, Ito and co-workers reported8g a novel
strategy for its stereoselective synthesis as a new entry
into the so-called intramolecular aglycon delivery
(IAD) approach. This strategy was subsequently ap-
plied to the completely stereocontrolled synthesis of the
core structure of Asn-linked glycoprotein oligosaccha-
rides.9c,d Herein we report on the application of this
method to the total synthesis of the glycosphingolipid.
The oligosaccharides of two glycolipids were the target
of the synthetic studies described herein as part of our
investigation into synthetic oligosaccharides of struc-
tural and biological interest.
deoxy-2-phthalimido-b-
-glucopyranoside (6),11 which
D
was prepared from the known phthalimido com-
pound.12 3-O-Levulinoylation13 of 6 with levulinic acid,
dicyclohexylcarbodiimide (DCC), and 4-(dimethy-
lamino)pyridine (DMAP) afforded compound 7. For
selective removal of the 2-(trimethylsilyl)ethyl (SE)
group, 7 was treated11 with trifluoroacetic acid in
dichloromethane for 1 h at 0 °C to give the 1-hydroxy
compound, which, on further treatment14 with
trichloroacetonitrile in the presence of 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) in dichloromethane for 2 h
at 0 °C, gave the corresponding trichloroacetimidate 8.
Syntheses of the target oligosaccharide.—b-Mannosy-
lation was conducted in a standard manner. Thus,
treatment of gluco-derived acceptor 911 with 5 and
2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in
,
dichloromethane in the presence of 4 A molecular
,
sieves (4 A MS) afforded the acetal. IAD was effected
by methyl trifluoromethanesulfonate (MeOTf) and 2,6-
di-tert-butyl-4-methylpyridine (DTBMP) in dichloro-
methane to afford a 37% yield of disaccharide, b-
mannopyranosyl-(1“4)-b- -glucopyranoside deriva-
tive 10. The b- configuration of the newly formed
D-
D
2. Results and discussion
D
glycosidic bond was indicated by the JC1,H1 value of
159.3 Hz in the 13C NMR spectrum15 (Table 2). Ben-
zoylation of 10 and subsequent removal of the 3%-O-TB-
DPS group from 11 by Bu4NF gave the disac-
Syntheses of monosaccharide deri6ati6es.—Syntheses
of the mannopyranosyl and 2-acetamido-2-deoxy-
D-
glucopyranosyl building blocks 5 and 8 were carried

I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
2039
the
corresponding
a-trichloroacetimidate
16.
charide acceptor 12 (Scheme 2). Glycosylation of 12
with Lemieux’s (phthalimido) donor 1312 in the pres-
Glycosylation16 of (2S,3R,4E)-2-azido-3-O-benzoyl-4-
octadecene-1,3-diol17 with the glycosyl donor 16, which
was carried out in the presence of TMSOTf and
AW300 MS for 8 h at 0 °C, afforded the desired
b-glycoside 17 (48%). Selective reduction18 of the azido
group in 17 with triphenylphosphine in 20:1 benzene–
water gave the amine, which on condensation with
stearic acid using 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (water soluble carbodi-
imide, WSC) in dichloromethane, gave the fully
protected derivative 18 (72%). Finally, removal of acyl
groups in 18 under basic conditions and column chro-
matography on Sephadex LH-20 furnished target gly-
,
ence of silver triflate (AgOTf) and 4 A MS in
dichloromethane for 24 h at rt gave the desired trisac-
charide 14 (72%), as evidenced by 1H NMR spec-
troscopy (H-1%%, 5.57 ppm, J 8.6 Hz). After removal of
the benzylidene and benzyl group with 10% Pd–C, the
phthalimido and O-benzoyl groups were removed by
refluxing with hydrazine hydrate in ethanol, giving free
amino and hydroxyl groups, which were subsequently
N- and O-acetylated with acetic anhydride and pyridine
giving 15. Selective removal of the SE group with
trifluoroacetic acid in dichloromethane and treatment
with trichloroacetonitrile in the presence of DBU gave
Table 2
13C NMR data (l) for selected compounds
Carbon atom
Compound
10
11
102.9
(159.4)
82.3
84.6
78.0
74.5
69.1
100.1
(155.2)
71.6
72.2
79.0
12
103.0
(157.2)
82.3
84.6
78.2
74.8
69.6
100.4
(157.2)
71.6
70.2
79.1
14
102.4
19
105.7
(159.3)
82.4
84.6
77.9
74.5
68.4
100.6
(159.7)
69.1
75.4
76.6
68.6
69.0
96.2
(165.5)
55.3
20
102.9
22
C-1 (Glc)
(JC,H)
2
3
4
5
6
C-1 (Man)
(JC,H)
2
3
4
5
6
C-1 (GlcNAc)
(JC,H)
2
3
4
5
6
C-1 (GalNAc)
2
3
4
5
6
102.9
(159.3)
82.3
102.8
(159.3)
82.2
84.6
78.0
74.6
68.1
99.9
(157.2)
69.6
77.2
75.6
69.5
69.4
97.1
(161.4)
54.5
70.8
69.0
71.7
69.0
(161.4)
82.2
84.5
78.4
74.6
68.9
99.9
(161.4)
69.3
75.0
75.6
68.5
69.3
96.9
(161.4)
55.1
73.5
71.3
73.2
71.3
82.3
84.6
77.9
74.5
68.6
99.8
84.1
77.8
74.3
68.4
100.7
(151.0)
71.4
68.9
75.4
75.2
67.8
68.1
96.7
72.7
78.4
66.6
68.6
66.8
68.5
67.7
68.6
55.1
73.9
74.7
74.1
70.3
95.7
52.0
72.6
73.5
70.5
67.4
67.3
18.3
−1.3
101.8
74.4
75.2
76.2
76.4
77.2
73.0
73.7
73.6
70.2
CH2CH2TMS
67.5
18.4
−1.4
101.7
749
67.3
18.5
−1.4
101.8
747
67.7
18.5
−1.4
102.4
74.3
67.3
18.3
−1.3
101.3
74.4
67.3
18.3
−1.3
101.2
74.4
74.8
74.9
75.6
76.7
67.3
18.4
−1.2
101.5
74.4
74.8
75.0
75.6
77.2
PhCHꢀ
PhCH2−
75.7
77.2
74.8
75.5
74.8
75.6
74.8
77.2

2040
I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
Scheme 2. (a) DDQ, CH2Cl2; (b) DTBMP, MeOTf, CH2Cl2; (c) BzCl, Pyr; (d) TBAF, AcOH, THF.
Scheme 3. (a) AgOTf, CH2Cl2; (b) (1) PdꢁC, AcOH, MeOH, (2) NH2NH2·H2O, EtOH, (3) Ac2O, Pyr; (c) (1) CF3COOH, CH2Cl2;
(2) CCl3CN, DBU, CH2Cl2; (d) (2S,3R,4E)-2-azido-3-benzoyl-4-octadecene-1,3-diol, TMSOTf, CH2Cl2; (e) (1) triphenylphos-
phine, benzene–water, (2) C17H35COOH, WSC, CH2Cl2; (f) NaOMe, 1,4-dioxane–MeOH.
colipid 1 (Scheme 3). The structure and purity of 1 was
demonstrated by the H NMR and MALDI-TOFMS
data. The synthesis of tetrasaccharide 2 began with the
TMSOTf-promoted coupling of 8 with 12, which af-
forded trisaccharide 19 (72%), as evidenced by 1H
NMR spectroscopy (H-1%% 5.34 ppm, J 8.4 Hz). Selec-
tive removal of the Lev group13 in 19 with hydrazine
acetate gave the partially protected compound 20.
Glycosylation19 of 20 with imidate 2120 in the presence
1
,
of TMSOTf and 4 A MS in dichloromethane for 24 h

I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
2041
was demonstrated by the MALDI-TOFMS data.
at rt gave the desired tetrasaccharide 22 (83%), as
evidenced by H NMR spectroscopy (H-1 of GalNAc,
1
In summary, the synthesis of oligosaccharides con-
taining the GlcNAcb(1“3)Man and GalNAcb(1“
4)GlcNAcb(1“3)Man structural motifs, has been
carried out. These structures may represent a new type
of glycolipid core of glycosphingolipids from the larvae
of the green-bottle fly, L. caesar.
5.43 ppm, J 8.5 Hz). Removal of the benzylidene and
benzyl groups with 10% Pd–C and then cleavage of the
phthalimido and the O-benzoyl groups with hydrazine
hydrate in ethanol afforded the fully deprotected
product, which was subsequently N- and O-acetylated
with acetic anhydride and pyridine to give 23. Selective
removal of the SE group, and treatment with
trichloroacetonitrile in the presence of DBU gave the
corresponding a-trichloroacetimidate 24. Glycosylation
of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-
diol with the glycosyl donor 24, which was carried out
in the presence of TMSOTf and AW300 MS for 6 h at
0 °C, afforded the desired b-glycoside 25 (40%). Selec-
tive reduction of the azido group in 25 with
triphenylphosphine in 20:1 benzene–water gave the
amine, which on condensation with stearic acid using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (WSC) in dichloromethane, gave the fully pro-
tected derivative 26 (67%). Finally, removal of the acyl
groups in 26 under the basic conditions and column
chromatography on Sephadex LH-20 furnished target
glycolipid 2 (Scheme 4). The structure and purity of 2
3. Experimental
General methods.—Optical rotations were measured
with a Jasco digital polarimeter. 1H and 13C NMR
spectra were recorded with a JMN A500 FT NMR
spectrometer with Me4Si as the internal standard for
solutions in CDCl3. MALDI-TOFMS was recorded on
a Perseptive Voyager RP mass spectrometer. High-reso-
lution mass spectra were recorded on a JEOL JMS-700
under FAB conditions. TLC was performed on Silica
Gel 60 F254 (E. Merck) with detection by quenching of
UV fluorescence and by charring with 10% H2SO4.
Column chromatography was carried out on Silica Gel
60 (E. Merck).
Ethyl
4,6-O-benzylidene-1-thio-a-D-mannopyran-
oside (3),10 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-b-
Scheme 4. (a) TMSOTf, CH2Cl2; (b) NH2NH2·AcOH, MeOH–THF; (c) (1) PdꢁC, AcOH, MeOH, (2) NH2NH2·H2O, EtOH, (3)
Ac2O, Pyr; (d) (1) CF3COOH, CH2Cl2; (2) CCl3CN, DBU, CH2Cl2; (e) (2S,3R,4E)-2-azido-3-benzoyl-4-octadecene-1,3-diol,
TMSOTf, CH2Cl2; (f) (1) triphenylphosphine, benzene–water, (2) C17H35COOH, WSC, CH2Cl2; (g) NaOMe, 1,4-dioxane–
MeOH.

2042
I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
D
-glucopyranoside (9),11 2-(trimethylsilyl)ethyl 3,6-di-
O-benzyl-2-deoxy-2-phthalimido-b- -glucopyranoside
(6),11
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl bromide (13),12 3,4,6-tri-O-acetyl-2-de-
(Na2SO4), and concentrated. The product was purified
D
by silica gel column chromatography using 2:1 hexane–
1
D-
AcOEt as eluent to give 7 (600 mg, 83%). H NMR
(CDCl3): l 7.68–6.87 (m, 14 H, 3 Ph), 5.15 (t, 1 H, J4,5
8.5 Hz, H-4), 5.13, 4.64, 4.60, 4.54 (each d, 4 H, Jgem
10.8 Hz, 2 PhCH2), 4.41 (t, 1 H, J2,3 10.4 Hz, H-2), 4.30
(d, 1 H, J1,2 8.3 Hz, H-1), 4.28 (t, 1 H, J3,4 8.5 Hz,
H-3), 3.92 (q, 1 H, OCH2CH2Si), 3.76 (q, 1 H, H-5),
3.65-3.63 (m, 2 H, H-6a, OCH2CH2Si), 3.46 (dd, 1 H,
J5,6a 16.7 Hz, J6a,6b 9.8 Hz, H-6b), 2.19 (s, 3 H, CH3),
1.57 (s, 4 H, CH2CH2), 0.83 (t, 2 H, OCH2CH2Si),
−0.08 (s, 9 H, Si(CH3)3). MALDI-TOFMS: Calcd for
C38H45NO9Si: m/z 687. Found: m/z 710 [M+Na]+.
HR FABMS: Calcd for C38H46NO9Si [M+H]+:
688.2941, Found 688.2923.
oxy-2-phthalimido-b-D-galactopyranosyl trichloroace-
timidate (21)20 and (2S,3R,4E)-2-azido-3-O-(benzoyl)-
4-octadecene-1,3-diol17 were prepared as reported in the
literature.
Ethyl
thio-h-
4,6-O-benzylidene-2-O-p-methoxybenzyl-1-
D-mannopyranoside (4).—Ethyl 4,6-O-benzyli-
dene-1-thio-a- -mannopyranoside (3: 500 mg, 1.60
D
mmol), Bu4NBr (153 mg, 0.50 mmol) and p-methoxy-
benzyl chloride (320 mL, 0.80 mmol) were dissolved in
CH2Cl2 (10 mL). Aq NaOH (1.0 mL of a 5% solution)
was added, and the mixture was stirred under reflux for
30 h. The reaction mixture was cooled, and the organic
layer was separated, washed with water, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 5:1 hexane–
AcOEt as eluent to give 4 (370 mg, 54%). [h]D +12.9°
3,6-Di-O-benzyl-2-deoxy-4-O-le6ulinoyl-2-phthal-
imido-i- -glucopyranosyl trichloroacetimidate (8).—To
D
a solution of 7 (292 mg, 0.43 mmol) in CH2Cl2 (5.0
mL), cooled to 0 °C was added CF3COOH (5.0 mL),
and the mixture was stirred for 1 h at rt and concen-
trated. AcOEt and toluene (1:2) were added and evapo-
rated to give the 1-hydroxy compound. To a solution of
the residue in CH2Cl2 (2.0 mL) cooled at 0 °C were
added CCl3CN (850 mL, 8.60 mmol) and DBU (64.0
mL, 0.43 mmol). The mixture was stirred for 30 min at
0 °C. After completion of the reaction, the mixture was
concentrated. Column chromatography of the residue
on silica gel (1:1 benzene–acetone) gave 8 (218 mg,
1
(c 3.0, CHCl3); H NMR (CDCl3): l 7.49–6.89 (m, 9
H, 2 Ph), 5.53 (s, 1 H, PhCH), 5.34 (s, 1 H, H-1),
4.67,4.56 (each d, 2 H, Jgem 11.6 Hz, PhCH2), 4.21–4.18
(m, 2 H, H-3, H-6a), 4.01 (q, 1 H, H-5), 3.91 (t, 1 H,
J4,5 9.2 Hz, H-4), 3.89 (d, 1 H, J2,3 9.7 Hz, H-2), 3.81
(t, 1 H, J6a,6b 11.0 Hz, H-6b), 3.78 (s, 3 H, PhOCH3),
2.60 (q, 2 H, SCH2CH3), 2.47 (s, 1 H, OH), 1.25 (t, 3 H,
SCH2CH3). MALDI-TOFMS: Calcd for C23H28O6S:
m/z 432. Found: m/z 455 [M+Na]+.
1
70%). H NMR (CDCl3): l 8.62 (s, 1 H, NH), 6.42 (d,
Ethyl 4,6-O-benzylidene-3-O-tert-butyldiphenylsilyl-
1 H, J1,2 8.6 Hz, H-1b), 6.36 (d, 1 H, J1,2 3.7 Hz, H-1a).
2 - O - p - methoxybenzyl -
1
- thio - h -
D
- mannopyranoside
2-(Trimethylsilyl)ethyl
butyldiphenylsilyl - i -
tri-O-benzyl-i-
4,6-O-benzylidene-3-O-tert-
(5).—To a solution of 4 (660 mg, 1.50 mmol) in DMF
(5.0 mL) were added imidazole (210 mg, 3.0 mmol) and
tert-butylchlorodiphenylsilane (0.80 mL, 3.0 mmol).
The reaction mixture was stirred for 18 h at 50 °C, then
extracted with AcOEt, washed with 5% HCl, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 8:1 hexane–
AcOEt as eluent to give 5 (934 mg, 91%). [h]D +14.2°
D
- mannopyranosyl - (1“4) - 2,3,6-
D
-glucopyranoside (10).—To a stirred
mixture of DDQ (68.1 mg, 0.30 mmol) and 4A MS in
dry CH2Cl2 (2.0 mL) were added 2-(trimethylsilyl)ethyl
2,3,6-tri-O-benzyl-b- -glucopyranoside (9, 105 mg, 0.20
D
mmol) and 5 (174 mg, 0.26 mmol) as a solution in dry
CH2Cl2 (5.0 mL) at 0 °C. The mixture was stirred for
10 min at 0 °C and for 1 h at rt. The resulting mixture
was quenched with a solution of ascorbic acid (0.7%)
and NaOH (0.9%) in water (3.0 mL), diluted with
AcOEt and filtered off. The filtrate was successively
washed with water, aq NaHCO3, dried (Na2SO4), and
concentrated to give mixed acetal. To a solution of this
acetal, 2,6-di-tert-butyl-4-methyl-pyridine (DTBMP:
225 mg, 1.10 mmol) and 4A MS in dry CH2Cl2 (5.0
mL) were stirred for 2 h at rt, then cooled to 0 °C.
Methyl trifuruoromethanesufonate (MeOTf, 113 mL,
5.0 mmol) was added to the mixture, which was stirred
for 10 min at 0 °C and for 24 h at rt, then neutralized
with Et3N. The mixture was filtered off and washed
with water, dried (Na2SO4), and concentrated. The
product was purified by silica gel column chromatogra-
phy using 10:1 hexane–AcOEt as eluent to give 10 (73.7
mg, 37%). [h]D −4.3° (c 1.0, CHCl3); 1H NMR
(CDCl3): l 7.66–7.17 (m, 30 H, 6 Ph), 5.35 (s, 1 H,
1
(c 0.7, CHCl3); H NMR (CDCl3): l 7.67–6.83 (19H,
m, 4 Ph), 5.41 (s, 1 H, PhCH), 5.01 (s, 1 H, H-1), 4.58,
4.40 (each d, 2 H, Jgem 11.8 Hz, PhCH2), 4.29 (dd, 1 H,
H-3), 4.14 (t, 1 H, J4,5 9.8 Hz, H-4), 4.11 (dd, 1 H,
H-6a), 4.02 (q, 1 H, H-5), 3.81–3.77 (m, 4 H, H-6b,
PhOCH3), 3.40 (d, 1 H, H-2), 2.47 (q, 2 H, SCH2CH3),
1.13 (t, 3 H, SCH2CH3), 1.05 (s, 9 H, Si(CH3)3).
MALDI-TOFMS: Calcd for C39H46O6SSi: m/z 670.
Found: m/z 693 [M+Na]+. HR FABMS: Calcd for
C39H47O6SSi [M+H]+: 671.2862, Found 671.2849.
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-
le6ulinoyl-2-phthalimido-i- -glucopyranoside (7).—To
D
a solution of 6 (630 mg, 1.10 mmol) in CH2Cl2 (5.0 mL)
was added levulinic acid (181 mL, 1.90 mmol), DCC
(407 mg, 2.0 mmol) and DMAP (36.0 mg, 0.30 mmol).
The reaction mixture was stirred for 2 h at rt, then
extracted with CHCl3, washed with 5% HCl, dried

I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
2043
PhCH), 4.95, 4.93, 4.81, 4.76, 4.70, 4.45 (each d, 6 H,
Jgem 10.9 Hz, 3 PhCH2), 4.37 (d, 1 H, J1,2 7.3 Hz, H-1),
4.22 (t, 1 H, J4,5 4.9 Hz, H-4%), 4.19 (s, 1 H, H-1%), 4.10
(dd, 1 H, H-6a), 3.99 (t, 1 H, J5,6b 9.2 Hz, H-6b), 3.93
(q, 1 H, OCH2CH2Si), 3.82 (dd, 1 H, J5,6b 9.2 Hz, J6a,6b
3.1 Hz, H-6a%), 3.78–3.76 (m, 2 H, H-2%, H-3%), 3.63 (t,
1 H, J3,4 8.5 Hz, H-3), 3.56 (q, 1 H, OCH2CH2Si),
3.51–3.48 (m, 2 H, H-5, H-6b%), 3.40–3.35 (m, 2 H,
H-2, H-4), 3.10 (t, 1 H, H-5%), 1.62 (s, 1 H, OH), 1.01
(s, 9 H, 3 CH3), 0.98 (t, 2 H, OCH2CH2Si), −0.08 (s,
J3,4 9.2 Hz, H-3), 3.44–3.36 (m, 2 H, H-5, H-5%), 3.27
(t, 1 H, J2,3 9.2 Hz, H-2), 0.92 (t, 2 H, OCH2CH2Si),
−0.08 (s, 9 H, Si(CH3)3). MALDI-TOFMS: Calcd for
C52H60O12Si: m/z 904. Found: m/z 927 [M+Na]+.
2-(Trimethylsilyl)ethyl 3,4,6,-tri-O-acetyl-2-deoxy-2-
phthalimido - i -
4,6-O-benzylidene-i-
tri-O-benzyl-i- -glucopyranoside (14).—A solution of
12 (140 mg, 0.15 mmol), 3,4,6-tri-O-acetyl-2-phthal-
imido-b- -glucopyranosyl bromide (13: 169 mg, 0.31
D
- glucopyranosyl - (1“3)-2-O-benzoyl-
D
-mannopyranosyl-(1“4)-2,3,6-
D
D
,
9
H, Si(CH3)3). MALDI-TOFMS: Calcd for
mmol) and 4 A MS in dry CH2Cl2 (2.0 mL) was stirred
for 2 h at rt, then cooled to −25 °C. Silver triflate
(AgOTf: 159 mg, 0.20 mmol) was added to the mixture,
which was stirred for 1 h at −25 °C and for 24 h at rt.
The insoluble materials were filtered off, and the filtrate
was washed with water, dried (Na2SO4), and concen-
trated. The product was purified by silica gel column
chromatography using 1:1 hexane–AcOEt as eluent to
C61H74O11Si2: m/z 1038. Found: m/z 1061 [M+Na]+.
HR FABMS: Calcd for C61H75O11Si2 [M+H]+:
1039.4847, Found 1039.4828.
2-(Trimethylsilyl)ethyl
dene-3-O-tert-butyldiphenylsilyl-i-
“4)-2,3,6-tri-O-benzyl-i- -glucopyranoside (11).—To
2-O-benzoyl-4,6-O-benzyli-
D-mannopyranosyl-(1
D
a solution of 10 (550 mg, 0.53 mmol) in pyridine (10
mL) were added benzoyl chloride (5.0 mL). The reac-
tion mixture was stirred for 5 h at rt, then extracted
with CHCl3, washed with 5% HCl, dried (Na2SO4), and
concentrated. The product was purified by silica gel
column chromatography using 10:1 hexane–AcOEt as
eluent to give 11 (540 mg, 89%). [h]D −9.3° (c 3.0,
1
give 14 (147 mg, 72%). [h]D −15.3° (c 0.2, CHCl3); H
NMR (CDCl3): l 8.08–7.14 (m, 29 H, 6 Ph), 5.64 (t, 1
H, J2%%,3%%=3%%,4%% 9.8 Hz, H-3%%), 5.59 (s, 1 H, PhCH), 5.57
(d, 1 H, J1,2 8.6 Hz, H-1%%), 5.46 (d, 1 H, J2%,3% 3.0 Hz,
H-2%), 4.86, 4.84, 4.73, 4.68, 4.62, 4.42 (each d, 6 H,
Jgem 11.2 Hz, 3 PhCH2), 4.53 (s, 1 H, H-1%), 4.35 (dd, 1
H, J5,6a 10.4 Hz, J6a,6b 4.9 Hz, H-6a%), 4.23 (d, 1 H, J1,2
7.9 Hz, H-1), 4.30–3.89 (m, 9 H, H-3%, H-4%, H-6a,
H-6b, H-6b% H-2%%, H-6a%%, H-6b%%, OCH2CH2Si,), 3.83
(q, 1 H, OCH2CH2Si), 3.77 (q, 1H, H-5), 3.57 (t, 1 H,
J3,4 9.2 Hz, H-3), 3.45–3.25 (m, 3 H, H-4, H-5%, H-5%%),
3.22 (t, 1 H, J2,3 8.5 Hz, H-2), 2.10, 2.08, 2.06 (each S,
9H, 3 Ac), 0.95 (t, 2 H, OCH2CH2Si), −0.08 (s, 9 H,
Si(CH3)3), 4.23 (d, 1 H, J1,2 7.9 Hz, H-1). MALDI-
TOFMS: Calcd for C72H79NO21Si: m/z 1321. Found:
m/z 1344 [M+Na]+. HR FABMS: Calcd for
C72H80NO21Si [M+H]+:1322.4991, Found 1322.4978.
2-(Trimethylsilyl)ethyl 2-acetamido-3,4,6,-tri-O-acet-
1
CHCl3); H NMR (CDCl3): l 8.17–6.97 (m, 35 H, 7
Ph), 5.55 (d, 1 H, H-2%), 5.18 (s, 1 H, PhCH), 4.90,
4.86, 4.76, 4.71, 4.64, 4.37 (each d, 6 H, Jgem 11.0 Hz, 3
PhCH2), 4.47 (s, 1 H, H-1%), 4.27 (d, 1 H, J1,2 7.9 Hz,
H-1), 4.22 (dd, 1 H, J5,6a 10.4 Hz, J6a,6b 4.9 Hz, H-6a%),
4.03 (dd, 1 H, J2,3 3.7 Hz, J3,4 9.8 Hz, H-3%), 3.98 (dd,
1 H, H-6a), 3.97 (t, 1 H, J4,5 9.2 Hz, H-4%), 3.94 (q, 1 H,
OCH2CH2Si), 3.79 (t, 1 H, J5,6b 10.4 Hz, H-6b%), 3.59–
3.50 (m, 3 H, H-3, H-6b, OCH2CH2Si), 3.35 (q, 1 H,
H-5), 3.32 (t, 1 H, J4,5 9.8 Hz, H-4), 3.26 (d, 1 H, H-2),
3.20 (q, 1 H, H-5%), 0.92 (t, 2 H, OCH2CH2Si), 0.82 (s,
9 H, 3 CH3), −0.08 (s, 9 H, Si(CH3)3). MALDI-
TOFMS: Calcd for C68H78O12Si2: m/z 1142. Found:
m/z 1165 [M+Na]+.
yl-2-deoxy - i -
acetyl-i- -mannopyranosyl-(1“4)-2,3,6-tri-O-acetyl-
i- -glucopyranoside (15).—A solution of 14 (147 mg,
D
- glucopyranosyl - (1“3) - 2,4,6 - tri-O-
D
2-(Trimethylsilyl)ethyl
dene-i- -mannopyranosyl-(1“4)-2,3,6-tri-O-benzyl-
i- -glucopyranoside (12).—To a solution of 11 (270
2-O-benzoyl-4,6-O-benzyli-
D
D
0.11 mmol) in MeOH (3.0 mL) and AcOH (10 mL) was
hydrogenolyzed in the presence of 10% Pd–C (140 mg)
for 24 h at rt, then filtered and concentrated. The
residue was dissolved in EtOH (5.0 mL), and hydrazine
monohydrate (5.0 mL) was added. The reaction mix-
ture was refluxed for 3 h, then concentrated. The
residue was acetylated with Ac2O (3.0 mL) in pyridine
(4.0 mL). The mixture was poured into ice-water and
extracted with CHCl3. The extract was washed sequen-
tially with 5% HCl, aq NaHCO3 and water, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 2:1 hexane–
AcOEt as eluent to give 15 (93.0 mg, 83%). [h]D
D
mg, 0.24 mmol) in THF (3.0 mL) were added t-Bu4NF
(TBAF, 1.0 M solution in THF 650 mL). The reaction
mixture was stirred for 1 h at rt, then extracted with
CHCl3, washed with 5% HCl, dried (Na2SO4), and
concentrated. The product was purified by silica gel
column chromatography using 1:1 hexane–AcOEt as
eluent to give 12 (140 mg, 80%). [h]D −20.5° (c 0.5,
1
CHCl3); H NMR (CDCl3): l 8.07–7.16 (m, 25 H, 5
Ph), 5.67 (d, 1 H, H-2%), 5.57 (s, 1 H, PhCH), 4.89,
4.85, 4.79, 4.74, 4.70, 4.65 (each d, 6 H, Jgem 11.0 Hz, 3
PhCH2), 4.76 (s, 1 H, H-1%), 4.35 (dd, 1 H, J5,6a 10.4
Hz, J6a,6b 4.9 Hz, H-6a%), 4.33 (d, 1 H, J1,2 7.9 Hz, H-1),
4.23 (t, 1 H, H-6a), 4.09 (dd, 1 H, J5,6a 9.7 Hz, J6a,6b 4.8
Hz, H-6b), 4.01–3.85 (m, 5 H, H-3%, OCH2CH2Si, H-4,
H-4%, H-6b%), 3.61 (q, 1 H, OCH2CH2Si), 3.57 (t, 1 H,
1
−88.1° (c 0.5, CHCl3); H NMR (CDCl3): l 5.63 (d, 1
H, NH) 5.39 (brs. 1 H, H-2%), 5.18–5.12 (m, 2 H, H-3,
H-3%%), 5.06 (t, 1 H, H-4%), 4.89–4.84 (m, 2 H, H-2,
H-4%%), 4.69 (d, 1 H, J1,2 8.5 Hz, H-1%%), 4.51 (s, 1 H,

2044
I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
H-1%), 4.46 (d, 1 H, J1,2 7.9 Hz, H-1), 2.14–2.10 (m,
30H, 10 Ac). MALDI-TOFMS: Calcd for C43H65NO25-
Si: m/z 1024. Found: m/z 1047 [M+Na]+.
aminopropyl)-3-ethylcarbodiimide
hydrochloride
(WSC, 12.8 mg, 0.06 mmol) in dry CH2Cl2 (1.0 mL) for
24 h at rt The mixture was diluted with CH2Cl2 (10
mL), washed with water, dried (Na2SO4), and concen-
trated. The product was purified by silica gel column
chromatography using 2:1 benzene–acetone as eluent
to give 18 (24.0 mg, 72%). [h]D −14.9° (c 0.5, CHCl3);
1H NMR (CDCl3): l 5.88–5.85 (m, 1 H, H-5 of
sphingosine), 5.55 (dd, 1 H, H-4 of sphingosine), 4.74
(d, 1 H, J1,2 8.5 Hz, H-1%%), 4.60 (s, 1 H, H-1%), 4.33 (d,
1 H, J1,2 7.9 Hz, H-1). MALDI-TOFMS: Calcd for
C81H126N2O28: m/z 1575. Found: m/z 1576 [M+H]+.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
pyranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -mannopyran-
osyl - (1“4) - 2,3,6 - tri - O - acetyl - h - - glucopyranosyl
D-gluco-
D
D
trichloroacetimidate (16).—To a solution of 15 (93.0
mg, 0.09 mmol) in CH2Cl2 (2.0 mL), cooled to 0 °C was
added CF3COOH (2.0 mL), and the mixture was stirred
for 30 min at rt and concentrated. AcOEt and toluene
(1:2) were added and evaporated to give the 1-hydroxy
compound. To a solution of the residue in CH2Cl2 (2.0
mL) cooled at 0 °C were added CCl3CN (325 mL, 3.20
mmol) and DBU (11.2 mL, 0.10 mmol). The mixture
was stirred for 30 min at 0 °C. After completion of the
reaction, the mixture was concentrated. Column chro-
matography of the residue on silica gel (1:1 hexane–
2-Acetamido-2-deoxy-i-
D-glucopyranosyl-(1“3)-i-
D-mannopyranosyl-(1“4)-i-D-glucopyranosyl-(1“1)-
(2S,3R,4E)-2-octadecamido-4-octadecene-1,3-diol (1).
—To a solution of 18 (24.0 mg, 0.02 mmol) in MeOH
(1.0 mL) and 1,4-dioxane (1.0 mL) was added NaOMe
(15.0 mg) at 40 °C, and the mixture was stirred for 20 h,
then neutralized with Amberlite IR-120 [H+]. The mix-
ture was filtered off and concentrated. The product was
purified by column chromatography (1:1 CHCl3–
MeOH) of the residue on Sephadex LH-20 to give 1
(12.5 mg, 82%). [h]D −11.4° (c 0.5, 1:1 CHCl3–
1
AcOEt) gave 16 (90.0 mg, 93%). H NMR (CDCl3): l
8.58 (s, 1 H, NH), 6.55 (d, 1H, J1,2 3.6 Hz, H-1), 4.71
(d, 1 H, J1,2 8.5 Hz, H-1%%), 4.49 (s, 1 H, H-1%). The
product was used directly in the following step.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
pyranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -mannopyran-
osyl-(1“4)-2,3,6-tri-O-acetyl-i- -glucopyranosyl-(1
D-gluco-
D
1
D
MeOH); H NMR (1:1 CDCl3–CD3OD): l 5.63–5.60
“1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-
1,3-diol (17).—To a solution of 16 (64.6 mg, 0.06
(m, 1 H, H-5 of sphingosine), 5.35 (dd, 1 H, H-4 of
sphingosine), 4.53 (d, 1 H, J1,2 8.5 Hz, H-1%%), 4.48 (s, 1
H, H-1%), 4.04 (d, 1 H, J1,2 7.6 Hz, H-1), 3.96 (t, 1 H,
H-3 of sphingosine). HRFABMS: Calcd for
C56H104N2NaO18 [M+Na]+: 1115.7182, Found
1115.7191.
mmol)
and
(2S,3R,4E)-2-azido-3-O-benzoyl-4-oc-
tadecene-1,3-diol (52.0 mg, 0.12 mmol) in dry CH2Cl2
(0.40 mL) was added AW300 MS (350 mg), and the
mixture was stirred for 12 h at rt, then cooled to 0 °C.
Trimethylsilyl trifluoromethanesulfonate (TMSOTf, 3.3
mL, 0.016 mmol) was added, and the mixture was
stirred for 8 h at 0 °C. The solids were filtered off and
washed with CH2Cl2. The combined filtrate and wash
were washed with NaHCO3 and water, dried (Na2SO4),
and concentrated. The product was purified by silica gel
column chromatography using 3:2 benzene–acetone as
eluent to give 17 (39.5 mg, 48%). [h]D −28.1° (c 0.9,
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-4-O-
le6ulinoyl-2-phthalimido-i-
O-benzoyl-4,6-O-benzylidene-i-
“4)-2,3,6-tri-O-benzyl-i- -glucopyranoside (19).—To
a solution of 12 (95.0 mg, 0.09 mmol), 8 (102 mg, 0.14
D-glucopyranosyl-(1“3)-2-
D
-mannopyranosyl-(1
D
,
mmol) and 4 A MS in dry CH2Cl2 (2.0 mL) were stirred
for 2 h at rt, then cooled to −25 °C. TMSOTf (3.1 mL,
0.02 mmol) was added to the mixture, which was stirred
for 1 h at −25 °C and for 24 h at rt. The mixture was
filtered, and the filtrate was washed with water, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 50:1 ben-
zene–acetone as eluent to give 19 (94.0 mg, 72%). [h]D
1
CHCl3); H NMR (CDCl3): l 5.94–5.90 (m, 1 H, H-5
of sphingosine), 5.68 (dd, 1 H, H-4 of sphingosine),
4.75 (d, 1 H, J1,2 8.5 Hz, H-1%%), 4.65 (s, 1 H, H-1%), 4.23
(d, 1 H, J1,2 7.9 Hz, H-1). MALDI-TOFMS: Calcd for
C63H90N4O27: m/z 1335. Found: m/z 1358 [M+Na]+.
HR FABMS: Calcd for C63H91N4O27 [M+H]+:
1335.5869, Found 1335.5832.
1
−9.1° (c 0.4, CHCl3); H NMR (CDCl3): l 7.51–6.84
(m, 39 H, 8 Ph), 5.60 (s, 1 H, PhCH), 5.43 (brs. 1 H,
H-2%), 5.34 (d, 1 H, J1,2 8.4 Hz, H-1%%), 5.10 (t, 1 H, J3%%,
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
pyranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -mannopyran-
osyl-(1“4)-2,3,6-tri-O-acetyl-i- -glucopyranosyl-(1
D-gluco-
D
9.7 Hz, H-4%%), 4.87, 4.80, 4.72, 4.67, 4.60, 4.50,
4%%=4%%, 5%%
D
4.45, 4.43, 4.40, 4.32 (each d, 10 H, 5 benzyl methyl-
ene), 4.41 (s, 1 H, H-1%), 4.34 (t, 1 H, H-3%%), 4.25 (d, 1
H, J1,2 7.9 Hz, H-1). 4.23 (t, 1 H, H-6a), 4.20 (t, 1 H,
H-2%%), 4.02 (dd, 1 H, H-3%), 3.94–3.82 (m, 4 H, H-6b,
4%, 6b, OCH2CH2), 3.75 (q, 1 H, H-5%%), 3.61 (dd, 1H,
H-6a%%), 3.54–3.46 (m, 4 H, H-3%, 6b%, 6b%%, OCH2CH2),
3.30–3.29 (m, 2 H, H-4, 5), 3.25–3.21 (m, 2 H, H-2,
5%), 2.09 (s, 3 H, CH3), 1.25 (s, 4 H, CH2CH2), 0.94 (t,
2 H, OCH2CH2Si), −0.03 (s, 9 H, Si(CH3)3). MALDI-
“1)-(2S,3R,4E)-3-O-benzoyl-2-octadecamido-4-octa-
decene-1,3-diol (18).—To an emulsion of 17 (30.0 mg,
0.02 mmol) in benzene (0.60 mL) and water (30.0 mL)
was added triphenylphosphine (17.6 mg, 0.06 mmol),
and the mixture was stirred for 24 h with the progress
of the reaction being monitored by TLC. The mixture
was concentrated, and the residue was stirred with
stearic acid (19.0 mg, 0.06 mmol) and 1-(3-dimethyl

I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
2045
TOFMS: Calcd for C85H91NO20Si: m/z 1474. Found:
m/z 1497[M+Na]+.
2-(Trimethylsilyl)ethyl 3,6-di-O-benzyl-2-deoxy-2-ph-
−0.03 (s, 9 H, Si(CH3)3). MALDI-TOFMS: Calcd for
C100H104N2O27Si: m/z 1793. Found: m/z 1816 [M+
Na]+. HRFABMS: Calcd for C100H105N2O27Si [M+
H]+: 1793.6673, Found 1793.6652.
thalimido-i-
O-benzylidene - i -
O-benzyl-i- -glucopyranoside (20).—To a solution of
D
-glucopyranosyl-(1“3)-2-O-benzoyl-4,6-
D
- mannopyranosyl - (1“4)-2,3,6-tri-
2-(Trimethylsilyl)ethyl 2-acetamido-3,4,6-tri-O-acet-
D
yl-2-deoxy - i -
ido-3,6-di-O-acetyl-2-deoxy-i-
3) - 2,4,6 - tri - O - acetyl - i - - mannopyranosyl-(1“4)-
2,3,6-tri-O-acetyl-i- -glucopyranoside (23). A solution
D
- glalactopyranosyl - (1“4)-2-acetam-
19 (94.0 mg, 0.63 mmol) in MeOH (0.3 mL) and THF
(3.0 mL) was added hydrazine acetate (24.0 mg, 2.6
mmol), and the mixture was stirred for 2 h at rt The
mixture was diluted with CH2Cl2 (10 mL), and the
organic layer was washed with NaHCO3 and water,
dried (Na2SO4), and concentrated. The product was
purified by silica gel column chromatography using 2:1
hexane–AcOEt as eluent to give 20 (80.0 mg, 91%).
[h]D −25.0° (c 0.3, CHCl3); 1H NMR (CDCl3): l
7.51–6.88 (m, 39 H, 8 Ph), 5.56 (s, 1 H, PhCH), 5.43
(brs. 1 H, H-2%), 5.34 (d, 1 H, J1,2 8.4 Hz, H-1%%), 4.87,
4.84, 4.73, 4.69, 4.65, 4.63, (each d, 6 H, 3 benzyl
methylene), 4.48–4.40 (m, 4 H, 2 benzyl methylene),
4.41 (s, 1 H, H-1%), 4.25 (d, 1 H, J1,2 7.9 Hz, H-1),
4.26–4.23 (m, 2 H, H-3%%, 6a), 4.18–4.07 (m, 2 H, H-2%%,
4%%), 3.98 (dd, 1 H, H-3%), 3.93–3.82 (m, 4 H, H-6b, 4%,
6b, OCH2CH2), 3.75 (q, 1 H, H-5%%), 3.61 (dd, 1H,
H-6a%%), 3.54-3.46 (m, 4 H, H-3, 6b%, 6b%%, OCH2CH2),
3.30–3.29 (m, 3 H, H-4, 5, 5%), 3.21 (m, 1 H, H-2), 3.02
(s, 1 H, OH), 0.98 (t, 2 H, OCH2CH2Si), −0.03 (s, 9
D
-glucopyranosyl-(1“
D
D
of 22 (76.0 mg, 0.04 mmol) in MeOH (3.0 mL) and
AcOH (1.0 mL) was hydrogenolyzed in the presence of
10% PdꢁC (90.0 mg) for 24 h at rt, then filtered and
concentrated. The residue and hydrazine monohydrate
(6.0 m L) were dissolved EtOH (8.0 mL). The reaction
mixture was refluxed for 3 h, then concentrated. The
residue was acetylated with Ac2O (3.0 mL) in pyridine
(4.0 mL). The mixture was poured into ice-water and
extracted with CHCl3. The extract was washed sequen-
tially with 5% HCl, aq NaHCO3 and water, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 2:1 ben-
zene–acetone as eluent to give 23 (47.6 mg, 83%). [h]D
1
−22.7° (c 1.0, CHCl3); H NMR (CDCl3): l 4.54 (d, 1
H, J1,2 8.3 Hz, H-1 of GlcNAc), 4.46 (d, 1 H, J1,2 8.5
Hz, H-1 of GalNAc), 4.30 (s, 1 H, H-1 of Man), 4.24
(d, 1 H, J1,2 7.9 Hz, H-1 of Glc). MALDI-TOFMS:
Calcd for C55H82N2O32Si: m/z 1311. Found: m/z 1334
[M+Na]+.
H,
Si(CH3)3).
MALDI-TOFMS:
Calcd
for
C80H85NO18Si: m/z 1376. Found: m/z 1399 [M+Na]+.
2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
topyranosyl-(1“4)-2-acetamido-3,6-di-O-acetyl-2-de-
oxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i-
-mannopyranosyl-(1“4)-2,3,6-tri-O-acetyl-h- -gluco-
D-glalac-
phthalimido - i -
benzyl-2-deoxy-2-phthalimido-i-
“3)-2-O-benzoyl-4,6-O-benzylidene-i-
osyl-(1“4)-2,3,6-tri-O-benzyl-i- -glucopyranoside
(22).—A solution of 20 (115 mg, 0.08 mmol), 3,4,6-tri-
O-acetyl-2-phthalimido-2-deoxy-b- -galactopyranosyl
trichloroacetimidate (21: 73.0 mg, 0.12 mmol) and MS
D
- glalactopyranosyl - (1“4)-3,6-di-O-
-glucopyranosyl-(1
-mannopyran-
D
D
D
D
D
D
pyranosyl tichloroacetimidate (24).—To a solution of 23
(47 mg, 0.04 mmol) in CH2Cl2 (1.0 mL), cooled to 0 °C
was added CF3COOH (1.0 mL), and the mixture was
stirred for 1 h at rt and concentrated. Ethyl acetate and
toluene (1:2) were added and evaporated to give the
1-hydroxy compound. To a solution of the residue in
CH2Cl2 (1.0 mL) cooled at 0 °C were added CCl3CN
(164 mL, 1.1 mmol) and DBU (5.50 mL, 0.04 mmol).
The mixture was stirred for 30 min at 0 °C. After
completion of the reaction, the mixture was concen-
trated. Column chromatography of the residue on silica
gel (1:1 benzene–acetone) gave 24 (47.0 mg, quantita-
D
,
4 A in dry CH2Cl2 (2.0 mL) was stirred for 2 h at rt,
then cooled to −25 °C. TMSOTf (3.0 mL, 0.24 mmol)
was added to the mixture, which was then stirred for 1
h at −25 °C and for 24 h at rt. The insoluble materials
were filtered off and the filtrate was washed with water,
dried (Na2SO4), and concentrated. The product was
purified by silica gel column chromatography using 2:1
hexane–AcOEt as eluent to give 22 (124 mg, 83%). [h]D
1
1
−34.1° (c 2.5 CHCl3); H NMR (CDCl3): l 7.92–6.88
tive). [h]D −3.3° (c 0.9, CHCl3). H NMR (CDCl3): l
(m, 43 H, 9 Ph), 5.71 (dd, 1 H, J2%%%,3%%% 11.5 Hz, J3%%%,4%%% 3.7
Hz, H-3%%%), 5.56 (s, 1 H, PhCH), 5.43 (d, 1 H, J1%%%, 2%%%
8.5 Hz, H-1%%%), 5.42 (br. s. 1 H, H-2%), 5.29 (d, 1 H,
H-4%%%), 5.20 (d, 1 H, J1,2 8.4 Hz, H-1%%), 4.87, 4.83, 4.76,
4.70, 4.63, 4.47, 4.41, 4.40 (m, 8 H, 4 benzyl methylene),
4.46 (t, 1 H, H-2%%%), 4.40 (s, 1 H, H-1%), 4.28 (dd, 1 H,
H-6a%), 4.21 (d, 1 H, J1,2 7.9 Hz, H-1), 4.26–4.08 (m,
6H, H-6a, 2%%, 4%%, 6a%%%, benzyl methylene), 3.98–3.80
(m, 6 H, H-4, 3%, 4%, 6b%, 6b%%, OCH2CH2), 3.55–3.21 (m,
10 H, H-2, 3, 5, 6b, 5%, 3%%, 5%%, 6a%%, 6b%%, OCH2CH2)
2.36–2.00 (m, 9 H, 3 Ac), 0.70 (t, 2 H, OCH2CH2Si),
8.84 (s, 1 H, NH), 6.47 (d, 1 H, J1,2 3.7 Hz, H-1 of Glc)
4.71 (d, 1 H, J1,2 8.4 Hz, H-1 of GalNAc), 4.62 (d, 1 H,
J1,2 8.4 Hz, H-1 of GlcNAc), 4.61 (s, 1 H, H-1 of Man).
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
pyranosyl-(1“4)-2-acetamido-3,6-di-O-acetyl-2-de-
oxy-i- -glucopyranosyl-(1“3)- 2,4,6-tri-O-acetyl-i-
- mannopyranosyl - (1“4) - 2,3,6 - tri - O - acetyl - i -
D-galacto-
D
D
D-
glucopyranosyl-(1“1)-(2S,3R,4E)-2-azido-3-O-ben-
zoyl-4-octadecene-1,3-diol (25).—To a solution of 24
(25.0 mg, 18.7 mmol) and (2S,3R,4E)-2-azido-3-O-ben-
zoyl-4-octadecene-1,3-diol (32.0 mg, 38.0 mmol) in dry

2046
I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
CH2Cl2 (0.40 mL) was added AW300 MS (350 mg),
and the mixture was stirred for 12 h at rt, then cooled
to 0 °C. TMSOTf (1.30 mL, 1.0 mmol) was added, and
the mixture was stirred for 6 h at 0 °C. The solids were
filtrated off and washed with CH2Cl2. The combined
filtrate and washings were washed with NaHCO3 and
water, dried (Na2SO4), and concentrated. The product
was purified by silica gel column chromatography using
1:1 benzene–acetone as eluent to give 25 (12.1 mg,
(s, 1 H, H-1 of Man), 4.41 (d, 1 H, J1,2 8.5 Hz, H-1 of
GalNAc), 4.18 (d, 1 H, J1,2 7.8 Hz, H-1 of Glc).
MALDI-TOFMS: Calcd for C64H117N3O23: m/z 1296.
Found: m/z 1319 [M+Na]+. HR-FABMS: Calcd for
C64H118N3O23 [M+H]+: 1296.8155, Found 1296.8134.
Acknowledgements
1
40%). [h]D −28.4° (c 0.2, CHCl3); H NMR (CDCl3):
This work was supported by a Grant-in-Aid for
Scientific Research (No. 12672062) from the Ministry
of Education, Science, Sports and Culture of Japan. We
gratefully acknowledge financial support in the form of
a Sasagawa Scientific Research Grant. The authors are
grateful to Ms. J. Hada for providing NMR and MS
data.
l 5.96–5.93 (m, 1 H, H-5 of sphingosine), 5.61 (dd, 1
H, H-4 of sphingosine), 4.68 (d, 1 H, J1,2 8.5 Hz, H-1
of GlcNAc), 4.41 (s, 1 H, H-1 of Man), 4.40 (d, 1 H,
J1,2 8.5 Hz, H-1 of GalNAc), 4.18 (d, 1 H, J1,2 7.8 Hz,
H-1
of
Glc).
MALDI-TOFMS:
Calcd
for
C75H107N5O34: m/z 1622. Found: m/z 1655 [M+Na]+.
HRFABMS: Calcd for C75H108N5O34 [M+H]+:
1622.6874, Found 1622.6849.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-i-
pyranosyl-(1“4)-2-acetamido-3,6-di-O-acetyl- 2-de-
oxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i-
- mannopyranosyl - (1“4) - 2,3,6 - tri - O - acetyl - i -
D
-galacto-
References
1. Varki, A. Glycobiology 1993, 3, 97–130.
2. Wiegandt, H. Glycolipids; Elsevier: New York, 1985.
3. (a) Hori, T.; Sugita, M.; Kanbayashi, J.; Itasaka, O. J.
Biochem. 1977, 81, 107–114;
D
D
D-
glucopyranosyl-(1“1)-(2S,3R,4E)-3-O-benzoyl-2-octa-
decamido-4-octadecene-1,3-diol (26).—To an emulsion
of 25 (7.6 mg, 4.6 mmol) in benzene (0.6 mL) and water
(30 mL) was added Ph3P (3.5 mg, 13.8 mmol), and the
mixture was stirred for 24 h with the progress of the
reaction being monitored by TLC. The mixture was
concentrated, and the residue was stirred with stearic
acid (2.5 mg, 14 mmol) and WSC (3.50 mg, 14 mmol) in
dry CH2Cl2 (0.6 mL) for 24 h at rt. The mixture was
diluted with CH2Cl2 (10 mL), washed with water, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography using 2:1 ben-
zene–acetone as eluent to give 26 (5.8 mg, 67%). [h]D
(b) Hori, T.; Takeda, H.; Sugita, M.; Itasaka, O. J.
Biochem. 1977, 82, 1281–1285;
(c) Hori, T.; Sugita, M.; Ando, S.; Kuwahara, M.; Ku-
mauchi, K.; Sugie, E.; Itasaka, O. J. Biol. Chem. 1981,
256, 10979–10985;
(d) Dennis, R. D.; Geyer, R.; Egge, H.; Menges, H.;
Stirm, S.; Wiegandt, H. Eur. J. Biochem. 1985, 146,
51–58.
4. (a) Takeda, T.; Hada, N.; Ogihara, Y. Chem. Pharm.
Bull. 1992, 40, 1930–1933;
(b) Takeda, T.; Hada, N.; Ogihara, Y. Chem. Pharm.
Bull. 1993, 41, 2058–2060;
(c) Hada, N.; Takeda, T.; Ogihara, Y. Carbohydr. Res.
1994, 258, 93–104;
(d) Hada, N.; Hayashi, E.; Takeda, T. Carbohydr. Res.
1999, 316, 58–70;
(e) Hada, N.; Matsuzaki, A.; Takeda, T. Chem. Pharm.
Bull. 1999, 47, 1265–1268;
(f) Hada, N.; Kuroda, M.; Takeda, T. Chem. Pharm.
Bull. 2000, 48, 1160–1165;
(g) Hada, N.; Ohtsuka, I.; Sugita, M.; Takeda, T. Tetra-
hedoron Lett. 2000, 41, 9065–9068.
1
−27.3° (c 0.13, CHCl3). H NMR (CDCl3): l 5.91–
5.90 (m, 1 H, H-5 of sphingosine), 5.52 (dd, 1 H, H-4
of sphingosine), 4.68 (d, 1 H, J1,2 8.5 Hz, H-1 of
GlcNAc), 4.41 (s, 1 H, H-1 of Man), 4.40 (d, 1 H, J1,2
8.5 Hz, H-1 of GalNAc), 4.20 (d, 1 H, J1,2 7.8 Hz, H-1
of Glc). MALDI-TOFMS: Calcd for C93H143N3O35:
m/z 1862. Found: m/z 1885 [M+Na]+.
5. Sugita, M.; Nishida, M.; Hori, T. J. Biochem. 1982, 92,
327–334.
6. Sugita, M.; Inagaki, F.; Naito, H.; Hori, T. J. Biochem.
1990, 107, 899–903.
2-Acetamido-2-deoxy-i-
2-acetamido-2-deoxy-i- -glucopyranosyl-(1“3)-i-
mannopyranosyl - (1“4) - i - - glucopyranosyl - (1“1)-
D-galactopyranosyl-(1“4)-
D
D-
D
7. Pozsgay, V. Stereoselective Synthesis of b-Mannoside. In
Carbohydrates in Chemistry and Biology; Ernst, B.; Hart,
G. W.; Sinay¨, P., Eds.; Wiley-VCH: Weinheim, 2000; Vol.
1, pp 319–344.
8. (a) Lichtenthaler, F. W.; Adams, T. S. J. Org. Chem.
1994, 59, 6728–6734;
(2S,3R,4E)-2-octadecamido-4-octadecene-1,3-diol (2).
—To a solution of 26 (5.8 mg, 3.10 mmol) in MeOH
(1.0 mL) and 1,4-dioxane (1.0 mL) was added NaOMe
(8.0 mg) at 40 °C and the mixture was stirred for 20 h,
then neutralized with Amberlite IR 120[H+]. The mix-
ture was filtered off and concentrated. The product was
purified by column chromatography (1:1 CHCl3–
MeOH) of the residue on Sephadex LH-20 giving 2 (3.0
(b) Sato, K.; Seki, H.; Yoshitomo, A.; Nanaumi, H.;
Takai, Y.; Ishido, Y. J. Carbohydr. Chem. 1998, 17,
703–727;
(c) Crich, D.; Sun, S. J. Am. Chem. Soc. 1998, 120,
435–436;
(d) Hodosi, G.; Kovac, P. J. Am. Chem. Soc. 1998, 119,
2335–2336;
1
mg, 74%). [h]D −12.7° (c 0.5, MeOH); H NMR (1:1
CDCl3–CD3OD): l 5.90–5.89 (m, 1 H, H-5 of sphin-
gosine), 4.68 (d, 1 H, J1,2 8.5 Hz, H-1 of GlcNAc), 4.42

I. Ohtsuka et al. / Carbohydrate Research 337 (2002) 2037–2047
2047
(e) Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994, 72,
1447–1465;
(f) Stork, G.; La Clair, J. J. J. Am. Chem. Soc. 1996, 118,
247–248;
12. Lemieux, R. U.; Takeda, T.; Chung, B. Y. ACS Symp.
Ser. 1976, 39, 90–115.
13. Kanie, O.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1996, 37,
4551–4554.
(g) Lergenmuller, M.; Nukada, T.; Kuramochi, K.; Dan,
A.; Ogawa, T.; Ito, Y. Eur. J. Org. Chem. 1999, 1367–
1376;
(h) Ohtake, H.; Ichiba, N.; Ikegami, S. J. Org. Chem.
2000, 65, 8171–8179.
14. Schmidt, R. R.; Grundler, G. Synthesis 1985, 885–
887.
15. Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2
1974, 293–297.
16. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212–235.
17. Kiso, M.; Nakamura, A.; Tomita, Y.; Hasegawa, A.
Carbohydr. Res. 1986, 158, 101–111.
18. (a) Ohtsubo, N.; Ishida, H.; Kiso, M.; Hasegawa, A.
Carbohydr. Res. 1998, 306, 517–530;
9. (a) Lichtenthaler, F. W.; Adams, T. S. J. Org. Chem.
1994, 59, 6735–6738;
(b) Crich, D.; Dai, Z. Tetrahedron 1999, 55, 1569–1580;
(c) Dan, A.; Ito, Y.; Ogawa, T. J. Org. Chem. 1995, 60,
4680–4681;
(d) Ohnishi, Y.; Ando, H.; Kawai, T.; Nakahara, Y.; Ito,
Y. Carbohydr. Res. 2000, 328, 263–276.
10. Garegg, P. J.; Kvarnstrom, I.; Niklasson, A.; Niklasson,
G.; Svensson, S. C. T. J. Carbohydr. Chem. 1993, 12,
933–953.
11. Jansson, K.; Ahlfors, S.; Kihlberg, T.; Magnusson, G. J.
Org. Chem. 1988, 53, 5629–5647.
(b) Gege, C.; Oscarson, S.; Schmidt, R. R. Tetrahedron
Lett. 2001, 42, 377–380.
19. Seifert, J.; Ogawa, T.; Ito, Y. Tetrahedron Lett. 1999, 40,
6803–6807.
20. van Seeventer, P. B.; Corsten, M. A.; Sanders, M. P.;
Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr. Res.
1997, 299, 171–179.