Synthesis and selectin-binding activity of N-deacetylsialyl Lewis X ganglioside

Article abstract of DOI:10.1016/S0008-6215(02)00274-4

A novel analogue of sialyl Lewis X ganglioside, N-deacetylsialyl Lewis X ganglioside, was synthesized. Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-α- D-galacto-2-nonulopyranosylonate-(2→3)-2,4,6-tri-O-benzoyl-D- galactopyranos

Full text of DOI:10.1016/S0008-6215(02)00274-4

Carbohydrate Research 337 (2002) 2111–2117
www.elsevier.com/locate/carres
Synthesis and selectin-binding activity of N-deacetylsialyl Lewis X
gangliosideꢀ
Masanori Yamaguchi,a Hideharu Ishida,a Christine Galustian,b Ten Feizi,b
Makoto Kisoa,*
aDepartment of Applied Bioorganic Chemistry, Gifu Uni6ersity, Gifu 501-1193, Japan
bThe Glycoscience Laboratory, Imperial College School of Medicine, Northwick Park Hospital, Watford Road, Harrow, Middlesex HA1 3UJ, UK
Received 11 April 2002; accepted 8 May 2002
Dedicated to Professor Derek Horton on the occasion of his 70th birthday
Abstract
A novel analogue of sialyl Lewis X ganglioside, N-deacetylsialyl Lewis X ganglioside, was synthesized. Methyl 4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-5-trifluoroacetamido-
ranosyl trichloroacetimidate was coupled with 2-(trimethylsilyl)ethyl [2-acetamido-6-O-benzyl-2-deoxy-3-O-(4-methoxybenzyl)-b-
-glucopyranosyl]-(1“3)-[2,4,6-tri-O-benzyl-b- -galactopyranosyl]-(1“4)-2,3,6-tri-O-benzyl-b- -galactopyranoside to give the
desired pentasaccharide in high yield. The glycosylation of the pentasaccharide acceptor, which was derived from its precursor by
removal of the 3-methoxybenzyl group, with the phenyl 1-thioglycoside derivative of -fucose using N-iodosuccinimide–tri-
D
-glycero-a-
D-galacto-2-nonulopyranosylonate-(2“3)-2,4,6-tri-O-benzoyl-D-galactopy-
D
D
D
L
fluoromethanesulfonic acid as promoter, produced the hexasaccharide. Proper manipulation of the protecting groups of the
hexasaccharide afforded the corresponding glycosyl imidate, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-oc-
tadecene-1,3-diol. Selective reduction of the azido group, N-acylation with octadecanoic acid, and the complete removal of the
protecting groups gave the desired N-deacetylsialyl Lewis X ganglioside. L-Selectin bound more strongly to N-deacetylsialyl Lewis
X ganglioside than to the sialyl Lewis X ganglioside, whereas E- and P-selectins bound equally well to the two gangliosides.
© 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Ganglioside; Sialyl Lewis X; Selectin; N-Deacetylsialic acid
1. Introduction
tion.4,5 We have demonstrated with chemically synthe-
sized gangliosides (Fig. 1) that the sialyl Lewis X (sLex)
sequence with 6-O-sulfo functionality at N-acetylglu-
cosamine (6-O-sulfo sLex)6 constitutes a strong ligand
It has been shown2,3 that selectin–carbohydrate in-
teractions mediate cell adhesions involved in various
biological processes. L-Selectin, a member of the se-
lectin family that is expressed on leukocytes, binds to
saccharide ligands on high endothelial cells in post-cap-
illary venules of lymph nodes. It plays a key role in the
initial stages of leukocyte extravasation into peripheral
lymph nodes and areas of acute and chronic inflamma-
ꢀ Synthetic studies on sialoglycoconjugates, Part 126. For
Part 125, see Ref. 1.
* Corresponding author. Tel./fax: +81-58-293-2916/2840
E-mail address: kiso@cc.gifu-u.ac.jp (M. Kiso).
Fig. 1. Synthetic analogs of sialyl Lewis X ganglioside.
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00274-4

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M. Yamaguchi et al. / Carbohydrate Research 337 (2002) 2111–2117
Scheme 2. Reagents and conditions: (a) (i) TFA, CH2Cl2, (ii)
CCl3CN, DBU, CH2Cl2, 0 °C; (b) TMSOTf, CH2Cl2, 0 °C; (c)
(i) H2S, pyr.–H2O, 0 °C, (ii) WSC, CH2Cl2; (d) MeONa,
MeOH, 45 °C, then H2O.
Scheme 1. Reagents and conditions: (a) H2, Pd–C, AcOH; (b)
Bz2O, DMAP, pyr.; (c) (i) TFA, CH2Cl2, (ii) CCl3CN, DBU,
CH2Cl2; (d) TMSOTf, CH2Cl2, 7 °C; (e) CAN, CH3CN–
H2O; (f) NIS–TfOH, benzene, 7°C; (g) (i) H2, Pd–C, EtOH,
HOAc, 40 °C, (ii) Ac2O, pyr., 40 °C.
ose derivative 6 in 78% yield. The p-methoxybenzyl
(MPM) group at C-3 of GlcNAc in 6 was selectively
removed (84%) by treatment with ceric ammonium
nitrate (CAN)–H2O, and the resulting 7 was fucosy-
lated with 86 in the presence of N-iodosuccinimide
(NIS)–trifluoromethanesulfonic acid (TfOH) in ben-
zene at 7 °C to produce the desired hexasaccharide 9 in
99% yield. Hydrogenolytic removal of the benzyl
groups in the hexasaccharide and the following O-
for L-selectin.7 Also, the N-deacetylated form of 6-O-
sulfo sLex (6-O-sulfo-N-deacetyl sLex)8,9 is a superior
ligand of L-selectin compared with the parental 6-O-
sulfo sLex.7,9 As a component of our continuing studies
on selectin ligands, in order to elucidate the structure
required for the recognition by L-selectin, we describe
the synthesis and selectin-binding activity of N-
deacetylsialyl Lewis X ganglioside (N-deacetyl sLex).
1
acetylation gave 10 in a quantitative yield. In the H
NMR spectrum of 10, a significant one-proton doublet
(J1,2 3.4 Hz, H-1 of fucose) appeared at l 5.07, showing
the newly formed glycoside to be an a-L-fucopyran-
oside. The hexasaccharide 10 was then converted to the
imidate derivative 11 by the removal of the 2-
(trimethylsilyl)ethyl group and the treatment of the
hemiacetal thus obtained with trichloroacetonitrile and
DBU (90% in two steps) (Schemes 1 and 2).
2. Results and discussion
For the synthesis of the target molecule, we used
methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroac-
Glycosylation of the azidosphingosine derivative
1211,12 with 11, and successive reduction of the azido
function and N-acylation was carried out using the
established method.12,13 Finally, removal of all protec-
tive groups under the basic conditions furnished the
target molecule, N-deacetylsialyl Lewis X (N-deacetyl
sLex) in 96% yield (Scheme 2).
A negative-ion mass spectrum acquired from the
target molecule gave an [M−H]− ion at m/z 1648 and
fragment ions at m/z 1399, 1237, 888 and 726. The ion
at m/z 1399 (−249 Da) corresponds to the fragment
obtained by glycosidic cleavage of the terminal N-
deacetylated neuraminic acid.
etamido-
ate - (2“3) - 2,4,6 - tri - O - benzoyl -
D
-glycero-a-
D
-galacto-2-nonulopyranosylon-
- galactopyranosyl-
D
trichloroacetimidate (4) as a glycosyl donor, which was
prepared from the known disaccharide 18,9 in 70% yield
(four steps). In the glycosyl donor 4, the amino func-
tion at C-5 of neuraminic acid is protected as the
trifluoroacetamide, which can be simultaneously con-
verted into the amino function again at the final O-
deacylation under the Zemple´n conditions.
Coupling of 4 with the suitably protected trisaccha-
ride acceptor 510 gave the sialyl a-(2“3)-neolactotetra-

M. Yamaguchi et al. / Carbohydrate Research 337 (2002) 2111–2117
2113
We investigated the binding of the selectins to the
target molecule, N-deacetyl sLex, in comparison with
sLex, 6-O-sulfo sLex, and 6-O-sulfo N-deacetyl sLex.
The results shown in Fig. 2 are one of three data sets
which were in overall agreement. L-Selectin consistently
bound more strongly to the N-deacetyl sLex than to
sLex, as in Fig. 2 panel B. The strength of binding to
the N-deacetyl sLex was of the same order as to the
6-O-sulfo sLex, but not as strong as to 6-O-sulfo N-
deacetyl sLex, which to date, is the most potent ligand
for L-selectin.7,9 Thus the hierarchy of L-selectin bind-
ing strengths was in the order 6-O-sulfo N-deacetyl
sLex\6-O-sulfo sLex=N-deacetyl sLex\sLex, show-
ing that N-deacetylation as well as 6-O-sulfation poten-
tiates the binding of L-selectin. With E-selectin, the
binding signals for the target compound and the three
other sLex analogs were consistently of the same order,
as shown in Fig. 2(A). With P-selectin, the binding to
the target compound was of the same order as that to
sLex and to 6-O-sulfo sLex. This binding was consis-
tently less than to the 6-O-sulfo N-deacetyl sLex.
specified solvent systems (v/v). Concentrations and
evaporations were conducted in vacuo.
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-
D
-glycero-h-
D-galac-
to-2-nonulopyranosylonate)-(2“3)-i-
D
-galactopyrano-
side (2).—A solution of 18,9 (1.55 g, 1.73 mmol) in
HOAc (100 mL) was vigorously stirred with 10% Pd–C
(1.5 g) for 12 h at room temperature (rt) under a H2
atmosphere. The catalyst was collected and washed
with MeOH. (Caution! Extreme fire hazard.) The com-
bined filtrate and washings were concentrated. Column
chromatography (20:1 CHCl3–MeOH) of the residue
on silica gel gave 2 (1.39 g, 96%) as an amorphous
1
mass: [h]D −9.3° (c 1.0, CHCl3). H NMR (CDCl3) l
1.09 (m, 2 H, Me3SiCH2CH2), 2.03, 2.04, 2.14, 2.15 (4
s, 12 H, 4 AcO), 1.99 (t, 1 H, Jgem=J3ax,4 13.4 Hz,
H-3IIax), 2.74 (dd, 1 H, J3eq,4 4.5 Hz, H-3IIeq), 3.85 (s,
3 H, COOMe), 4.89 (dd, 1 H, J8,9 1.8, Jgem 10.7 Hz,
H-9II), 4.43 (d, 1 H, J1,2 7.7 Hz, H-1I), 5.05 (m, 1 H,
H-4II), 5.28 (dd, 1 H, J6,7 1.8, J7,8 8.4 Hz, H-7II), 5.43
(m, 1 H, H-8II) 6.71 (d, 1 H, J5,NH 9.6 Hz, NHII). Anal.
Calcd for C31H48F3NO18Si (807.79): C, 46.09; H, 5.99;
N, 1.73. Found: C, 46.06; H, 5.85; N, 1.60.
3. Experimental
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-
to-2-nonulopyranosylonate)-(2“3)-2,4,6-tri-O-benzoyl-
i- -galactopyranoside (3).—A mixture of 2 (814 mg,
D-glycero-h-D-galac-
General methods.—Specific rotations were deter-
mined with a Horiba SEPA-300 high-sensitive polar-
imeter at 25 °C, and 1H NMR spectra were recorded on
Varian Unity Inova (400 and 500 MHz) spectrometers
with TMS as the internal standard. Liquid secondary-
ion mass spectrometry (LSIMS) was carried out on a
VG Analytical ZAB2-E mass spectrometer, fitted with a
cesium ion gun operated at 25 keV and an emission
current of 0.5 mA. Spectra were acquired in the nega-
tive-ion mode directly from the surface of a silica gel
TLC plate as described.14 Preparative thin-layer chro-
matography (TLC) was performed on Silica Gel 60 (E.
Merck), and column chromatography on Silica Gel
(Fuji Silysia Co., 300 mesh) was performed with the
D
1.01 mmol), benzoic anhydride (1.2 g, 5.28 mmol) and
4-dimethylaminopyridine (DMAP, 53 mg, 0.43 mmol)
was dissolved in pyridine (5 mL), and the mixture was
stirred for 12 h at rt, then cooled to 0 °C. MeOH (5
mL) was added and the mixture was concentrated. The
residue was extracted with CHCl3 and successively
washed with cold 2 M HCl and water, dried (Na2SO4)
and concentrated. Column chromatography (1:4
EtOAc–hexane) of the residue on silica gel resulted in 3
(1.06 g, 92%) as an amorphous mass: [h]D +26.4° (c
1.0, CHCl3). 1H NMR (CDCl3): l 0.93 (m, 2 H,
Me3SiCH2CH2), 1.44, 1.91, 2.06, 2.18 (4 s, 12 H, 4
Fig. 2. Comparison of the selectin binding siganls elicited by the largest compound, N-deacetyl sLex, with those elicited by sLex,
6-O-sulfo sLex and 6-O-sulfo-N-deacetyl sLex. Binding of the recomboinant soluble E-, L- and P-selectins (panels A, B, and C,
respectively) to the glycolipids immobialized in microwells was assayed as described in Section 2. Results are expressed as means
in duplicate wells. The range, where visible, is indicated as error bars.

2114
M. Yamaguchi et al. / Carbohydrate Research 337 (2002) 2111–2117
AcO), 1.67 (t, 1 H, Jgem=J3ax,4 12.8 Hz, H-3IIax), 2.53
(dd, 1 H, J3eq,4 4.5 Hz, H-3IIeq), 3.86 (s, 3 H, COOMe),
3.96 (dd, 1 H, Jgem 12.3, J8,9 4.3 Hz, H-9II), 4.33 (dd, 1
H, J8,9% 2.9 Hz, H-9%II), 4.35 (dd, 1 H, J5,6% 5.0, Jgem 9.8
Hz, H-6%I), 4.51 (dd, 1 H, J5,6 6.8 Hz, H-6I) 4.83 (d, 1 H,
J1,2 8.0 Hz, H-1I), 4.95 (m, 1 H, H-4II), 5.18 (dd, 1 H,
J6,7 2.0, J7,8 9.3 Hz, H-7II), 5.42 (dd, 1 H, J2,3 10.1 Hz,
H-2I), 5.62 (m, 1 H, H-8II), 6.26 (d, 1 H, J5,NH 9.1 Hz,
NHII), 7.26–8.18 (m, 15 H, 3 Ph). Anal. Calcd for
C52H60F3NO21Si (1120.12): C, 55.76; H, 5.40; N, 1.25.
Found: C, 55.67; H, 5.20; N, 1.07.
gave 6 (622 mg, 78%) as an amorphous mass: [h]D
+7.3° (c 0.38, CHCl3). H NMR (CDCl3) l 1.02 (m, 2
1
H, Me3SiCH2CH2), 1.44 (s, 3 H, AcN), 1.48, 1.90, 1.96,
2.16 (4 s, 12 H, 4 AcO), 1.67 (t, 1 H, Jgem=J3ax,4 12.0
Hz, H-3Vax), 2.49 (dd, 1 H, J3eq,4 4.3 Hz, H-3Veq), 3.64
(s, 3 H, MeOPh), 3.84 (s, 3 H, COOMe), 4.89 (d, 1 H,
J1,2 8.7 Hz, H-1IV), 5.67 (m, 1 H, H-8V), 6.23 (d, 1 H,
J5,NH 9.1 Hz, NHV), 6.60–8.23 (m, 54 H, aromatic
protons). Anal. Calcd for C128H141F3N2O37Si (2382.89):
C, 64.47; H, 5.96; N, 1.17. Found: C, 64.47; H, 5.89; N,
1.02.
Methyl
fluoroacetamido-
sylonate-(2“3)-2,4,6-tri-O-benzoyl-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-tri-
-glycero-h- -galacto-2-nonulopyrano-
-galactopyran-
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
D
D
3,5-dideoxy-5-trifluoroacetamido-
to-2-nonulopyranosylonate)-(2“3)-2,4,6-tri-O-benzoyl-
i- -galactopyranosyl-(1“4)-2-acetamido-6-O-benzyl-
2-deoxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-O-ben-
zyl-i- -galactopyranosyl-(1“4)-2,3,6-tri-O-benzyl-i-
-glucopyranoside (7).—To a solution of 6 (331 mg,
D-glycero-h-D-galac-
D
osyltrichloroacetimidate (4).—Compound 3 (1.06 g,
0.99 mmol) was treated with trifluoroacetic acid (4.4
mL) in CH2Cl2 (7 mL) for 1 h at rt. EtOAc (5 mL) was
added, and the mixture was concentrated. Column
chromatography (1:2 EtOAc–hexane) of the residue on
silica gel gave the free 1-OH derivative (898 mg). This
compound (898 mg, 0.91 mmol) was treated with
Cl3CCN (2.8 mL, 28 mmol) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU; 144 mL, 0.96 mmol) in
CH2Cl2 (9 mL) for 2 h at 0 °C. The mixture was
concentrated, and the residue was chromatographed
(100:1 CHCl3–MeOH) on a column of silica gel to give
the trichloroacetimidate 4 as the mixture of a and b
D
D
D
D
0.14 mmol) in CH3CN (3.6 mL) and water (0.4 mL)
was added ceric ammonium nitrate (CAN, 300 mg, 0.55
mmol), and the mixture was stirred for 1 h at rt and
extracted with CHCl3. The extract was successively
washed with 1 M Na2CO3 and water, dried (Na2SO4)
and concentrated. Column chromatography (80:1
CHCl3–MeOH) of the residue on silica gel gave 7 (263
mg, 84%) as an amorphous mass: [h]D −7.0° (c 0.5,
CHCl3). 1H NMR (CDCl3):
l 1.02 (m, 2 H,
1
isomers (a:b=2:1) (1.02 g, 92% two steps). H NMR
Me3SiCH2CH2), 1.44 (s, 3 H, AcN), 1.48, 1.90, 1.96,
2.16 (4 s, 12 H, 4 AcO), 1.70 (dd, 1 H, Jgem 12.4, J3ax,4
12.0 Hz, H-3Vax), 2.49 (dd, 1 H, J3eq,4 4.3 Hz, H-3Veq),
3.84 (s, 3 H, COOMe), 5.67 (m, 1 H, H-8V), 7.08–8.23
(m, 50 H, 10 Ph). Anal. Calcd for C120H133F3N2O36Si
(2264.44): C, 63.65; H, 5.92; N, 1.24. Found: C, 63.56;
H, 5.81; N, 1.23.
(CDCl3) of a isomer: l 1.91, 1.95, 2.07, 2.10 (4 s, 12 H,
4 AcO), 1.63 (t, 1 H, Jgem=J3ax,4 12.8 Hz, H-3Vax),
2.56 (dd, 1 H, Jgem 12.8, J3eq,4 4.4 Hz, H-3Veq), 3.81 (s,
3 H, COOMe), 5.01 (m, 1 H, H-4II), 5.43 (dd, 1 H, J6,7
1.5, J7,8 9.2 Hz, H-7II), 5.60 (m, 1 H, H-8II), 6.54 (d, 1
H, J5,NH 9.5 Hz, NHII), 6.87 (d, 1 H, J1,2 3.7 Hz, H-1I),
7.27–8.19 (m, 15 H, 3 Ph), 8.64 (s, 1 H, NH of
imidate). Anal. Calcd for C49H48Cl3F3N2O21 (1164.27):
C, 50.55; H, 4.16; N, 2.41. Found: C, 50.49; H, 3.96; N,
2.25.
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-
to-2-nonulopyranosylonate)-(2“3)-2,4,6-tri-O-benzoyl-
i- -galactopyranosyl-(1“4)-[2,3,4-tri-O-benzyl-h-
fucopyranosyl-(1“3)]-2-acetamido-6-O-benzyl-2-de-
oxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-O-benzyl-i-
- galactopyranosyl - (1“4) - 2,3,6 - tri - O - benzyl - i -
D-glycero-h-D-galac-
D
L-
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-
to-2-nonulopyranosylonate)-(2“3)-2,4,6-tri-O-benzoyl-
i- -galactopyranosyl-(1“4)-2-acetamido-6-O-benzyl-
2-deoxy-3-O - p - methoxybenzyl - i - - glucopyranosyl -
(3)-2,4,6-tri-O-benzyl-i- -galactopyranosyl-(1“4)-
2,3,6-tri-O-benzyl-i- -glucopyranoside (6).—To a so-
D
-glycero-h-
D
-galac-
D
D
D-
D
glucopyranoside (9).—To a solution of 7 (263 mg, 0.12
D
mmol) and 86 (74 mg, 0.20 mmol) in dry benzene (6
,
D
mL) was added 4 A molecular sieves (400 mg), and the
D
mixture was stirred for 2 h at rt, then cooled to 0 °C.
N-Iodosuccinimide (NIS; 136 mg, 0.6 mmol) and trifl-
uoromethanesulfonic acid (TfOH, 8.9 mL, 0.1 mmol)
were added to the mixture, and the resultant mixture
was stirred for 2 h at 7 °C and neutralized with Et3N.
After dilution with CHCl3, the precipitate was filtered
off and washed with CHCl3. The filtrate and washings
were combined, and successively washed with 1 M aq
Na2CO3 and satd aq Na2S2O3, dried (Na2SO4) and
concentrated. Column chromatography (80:1 CHCl3–
MeOH) of the residue on silica gel afforded 9 (298 mg,
99%) as an amorphous mass: [h]D −8.1° (c 1.2,
lution of 4 (850 mg, 0.77 mmol) and the trisaccharide
acceptor 510 (540 mg, 0.39 mmol) in dry CH2Cl2 (8 mL)
,
was added 4 A molecular sieves (800 mg), and the
mixture was stirred for 3 h at rt, then cooled to 0 °C.
Trimethylsilyl trifluoromethanesulfonate (TMSOTf;
13.5 mL, 67.5 mmol) was added to the mixture, and
stirring was continued for 12 h at 7 °C. The reaction
mixture was neutralized with Et3N and filtered, and the
residue was washed with CHCl3. The combined filtrate
and washings were concentrated. Column chromatogra-
phy (100:1 CHCl3–MeOH) of the residue on silica gel

M. Yamaguchi et al. / Carbohydrate Research 337 (2002) 2111–2117
2115
CHCl3). 1H NMR (CDCl3):
l
0.99 (m,
2
H,
CH2Cl2 (4 mL) for 3 h at rt. EtOAc (1 mL) was added
and the mixture was concentrated. Column chromatog-
raphy (50:1 CHCl3–MeOH) of the residue on silica gel
gave the 1-OH free derivative. This compound was
treated with Cl3CCN (195 mL, 15.6 mmol) and 1,8-diaz-
abicyclo[5.4.0]undec-7-ene (DBU, 9.5 mL, 0.059 mmol)
in CH2Cl2 (4 mL) for 2 h at 0 °C. The mixture was
concentrated, and the residue was chromatographed
(40:1 CHCl3–MeOH) on a column of silica gel to give
the trichloroacetimidate 11 (246 mg, 90% two steps) as
Me3SiCH2CH2), 1.11 (d, 3 H, J5,6 6.4 Hz, H-6VI), 1.49
(s, 3 H, AcN), 1.52, 1.94, 1.99, 2.18 (4 s, 12 H, 4 AcO),
1.71 (t, 1 H, Jgem=J3eq,4 12.3 Hz, H-3Vax), 2.51 (dd, 1
H, J3eq,4 4.3 Hz, H-3Veq), 3.83 (s, 3 H, COOMe), 4.32
(d, 1 H, J1,2 7.7 Hz, H-1I), 4.43 (m, 1 H, H-5VI), 4.81 (d,
1 H, J1,2 8.2 Hz, H-1IV), 5.04 (d, 1 H, J1,2 3.2 Hz,
H-1VI), 5.25 (dd, 1 H, J6,7 2.0, J7,8 9.6 Hz, H-7V), 5.27
(d, 1 H, J2,NH 9.6 Hz, NHIII), 5.69 (m, 1 H, H-8V), 6.17
(d, 1 H, J5,NH 8.9 Hz, NHV), 7.17–8.23 (m, 65 H, 13
Ph). Anal. Calcd for C147H161F3N2O40Si (2680.96): C,
65.86; H, 6.05; N, 1.04. Found: C, 65.64; H, 6.03; N,
1.01.
1
an amorphous mass: [h]D −10.8° (c 0.1, CHCl3); H
NMR (CDCl3): l 1.23 (d, 3 H, J5,6 6.4 Hz, H-6VI), 1.55
(s, 3 H, AcN), 1.63 (dd, 1 H, Jgem 12.8, J3ax,4 12.0 Hz,
H-3Vax), 1.83–2.11 (s, 42 H, 14 AcO), 2.56 (dd, 1 H,
Jgem 12.8, J3eq,4 4.3 Hz, H-3Veq), 3.84 (s, 3 H, COOMe),
4.68 (dd, J1,2 3.4, J2,3 6.3 Hz, H-2VI), 4.81 (m, 1 H,
H-5VI), 5.06 (d, 1 H, H-1VI), 5.66 (m, 1 H, H-8V), 6.08
(d, 1 H, J5,NH 9.1 Hz, NHV), 6.47 (d, 1 H, J1,2 3.7 Hz,
H-1I), 7.45–8.19 (m, 15 H, 3 Ph), 8.53 (s, 1 H, CꢀNH).
Anal. Calcd for C95H111Cl3F3N3O50 (2258.26): C, 50.53;
H, 4.95; N, 1.86. Found: C, 50.42; H, 4.95; N, 1.84.
2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-trifluoroacetamido-
to-2-nonulopyranosylonate)-(2“3)-2,4,6-tri-O-benzoyl-
i- -galactopyranosyl-(1“4)-[2,3,4-tri-O-acetyl-h-
fucopyranosyl-(1“3)]-2-acetamido-6-O-acetyl-2-de-
oxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i-
- galactopyranosyl - (1“4) - 2,3,6 - tri - O - acetyl - i - D-
D-glycero-h-D-galac-
D
L-
D
D
glucopyranoside (10).—A solution of 9 (298 mg, 0.12
mmol) in EtOH (40 mL) and AcOH (8 mL) was
vigorously stirred in the presence of 10% Pd–C (300
mg) for 48 h at 40 °C under an H2 atmosphere. The
catalyst was collected and washed with MeOH. (Cau-
tion! Extreme fire hazard.) The combined filtrate and
washings were concentrated, and the residue was
treated with Ac2O (5.5 mL) and pyridine (9 mL) for 24
h at 40 °C. MeOH (10 mL) was added at 0 °C, and the
mixture was concentrated. The residue was extracted
with CHCl3 and successively washed with cold 2 M
HCl and water, dried (Na2SO4) and concentrated.
Column chromatography (50:1 CHCl3–MeOH) of the
residue on silica gel gave 10 (250 mg, quant) as an
(Methyl
fluoroacetamido-
osylonate) - (2“3) - 2,4,6 - tri - O - benzoyl - i -
pyranosyl-(1“4)-[2,3,4-tri-O-acetyl-h- -fucopyran-
osyl-(1“3)]-2-acetamido-6-O-acetyl-2-deoxy-i-
glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -galacto-
pyranosyl-(1“4)-2,3,6-tri-O-acetyl-i- -glucopyran-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-tri-
-glycero-h- -galacto-2-nonulopyran-
- galacto-
D
D
D
L
D-
D
D
osyl-(1“1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octa-
decene-1,3-diol (13).—To a solution of 11 (98 mg, 46
mmol)
and
(2S,3R,4E)-2-azido-3-O-benzol-4-oc-
tadecene-1,3-diol (12; 29 mg, 67 mmol) in dry
,
dichloromethane (0.5 mL) was added 4 A molecular
sieves (type AW300; 300 mg), and the mixture was
stirred for 2 h at rt, and then cooled to 0 °C.
Trimethylsilyl trifluoromethanesulfonate (TMSOTf; 50
mM in CH2Cl2, 86 mL, 4.3 mmol) was added to the
mixture, and this was stirred for 24 h at 0 °C, neutral-
ized with Et3N filtered and concentrated. The residue
was dissolved in 80% aq HOAc (3 mL), stirred for 2 h
at rt and then concentrated. Chromatography (60:1
CHCl3–MeOH) of the residue on silica gel afforded 13
(24 mg, 21%) as an amorphous mass: [h]D −15.2° (c
1
amorphous mass: [h]D −13.3°(c 0.2, CHCl3). H NMR
(CDCl3): l 0.98 (m, 2 H, Me3SiCH2CH2), 1.22 (d, 3 H,
J5,6 6.4 Hz, H-6VI), 1.56 (s, 3 H, AcN), 1.65 (dd, 1 H,
Jgem 12.4, J3ax,4 12.0 Hz, H-3Vax), 1.84–2.13 (14 s, 42
H, 14 AcO), 2.45 (dd, 1 H, J3eq,4 4.3 Hz, H-3Veq), 3.83
(s, 3 H, COOMe), 4.44 (d, 1 H, J1,2 7.7 Hz, H-1I), 4.68
(dd, J1,2 3.4, J2,3 6.3 Hz, H-2VI), 4.81 (m, 1 H, H-5VI),
4.84 (dd, J1,2 7.7, J2,3 9.6 Hz, H-2I), 5.06 (d, 1 H,
H-1VI), 5.22 (d, 1 H, J2,NH 9.8 Hz, NHIII), 5.27 (dd, 1
H, J6,7 3.4, J7,8 7.5 Hz, H-7V), 5.59 (m, 1 H, H-8V), 6.45
(d, 1 H, J5,NH 9.1 Hz, NHV), 7.49–8.18 (m, 15 H, 3 Ph).
Anal. Calcd for C98H123F3N2O50Si (2214.11): C, 53.16;
H, 5.60; N, 1.27. Found: C, 52.98; H, 5.42; N, 1.17.
1
0.4, CHCl3). H NMR (CDCl3): l 0.87 (t, 3 H, JMe,CH2
7.0 Hz, MeCH2), 1.26 (s, 22 H, 11 CH2), 1.61 (s, 3 H,
AcN), 1.67 (dd, 1 H, Jgem 12.6, J3ax,4 12.3 Hz, H-3Vax),
1.83–2.12 (14 s, 42 H, 14 AcO), 2.51 (dd, 1 H, J3eq,4 4.6
Hz, H-3Veq), 3.84 (s, 3 H, COOMe), 4.47 (d, 1 H, J1,2
7.7 Hz, H-1I), 4.72 (dd, J1,2 3.4, J2,3 6.3 Hz, H-2VI), 4.82
(d, 1 H, J1,2 8.2 Hz, H-1IV), 5.07 (d, 1 H, H-1VI), 5.24
(dd, 1 H, J6,7 3.8, J7,8 9.6 Hz, H-7V), 5.66 (m, 1 H,
H-8V), 5.93 (dt, 1 H, J4,5 14.2, J5,6=J5%,6 7.3 Hz, H-5 of
sphingosine), 6.12 (d, 1 H, J5,NH 8.6 Hz, NHV), 7.45–
8.17 (m, 20 H, 4 Ph). Anal. Calcd for C118H148F3N5O52
(2525.46): C, 56.12; H, 5.91; N, 2.77. Found: C, 55.86;
H, 5.79; N, 2.54.
(Methyl
fluoroacetamido-
osylonate) - (2“3) - 2,4,6 - tri - O - benzoyl - i -
pyranosyl-(1“4)-[2,3,4-tri-O-acetyl-h- -fucopyran-
osyl-(1“3)]-2-acetamido-6-O-acetyl-2-deoxy-i-
glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -galacto-
pyranosyl-(1“4)-2,3,6-tri-O-acetyl-h- -glucopyranosyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-tri-
-glycero-h- -galacto-2-nonulopyran-
- galacto-
D
D
D
L
D-
D
D
trichloroacetimidate (11).—Compound 10 (250 mg, 0.12
mmol) was treated with trifluoroacetic acid (2 mL) in

2116
M. Yamaguchi et al. / Carbohydrate Research 337 (2002) 2111–2117
(Methyl
fluoroacetamido-
osylonate)-(2“3)-2,4,6-tri-O-benzoyl-i-
ranosyl-(1“4)-[2,3,4-tri-O-acetyl-h- -fucopyranosyl-
(1“3)]-2-acetamido-6-O-acetyl-2-deoxy-i- -glucopy-
ranosyl-(1“3)-2,4,6-tri-O-acetyl-i- -galactopyran-
osyl-(1“4)-2,3,6-tri-O-acetyl-i- -glucopyranosyl-(1“
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-tri-
-glycero-h- -galacto-2-nonulopyran-
-galactopy-
J3eq,4 3.2 Hz, H-3Veq), 4.29, 4.35, 4.86 (3 d, 3 H, J1,2
D
D
7.3, 7.5, 7.3 Hz, for each three b-anomeric-H), 5.05 (d,
1 H, J1,2 4.1 Hz, H-1VI), 5.44 (m, 1 H, H-4 of sphin-
gosine), 5.67 (m, 1 H, H-5 of sphingosine). Anal. Calcd
for C77H139F3N3O34 (1650.95) C, 56.02; H, 8.49; N,
2.55. Found: C, 55.94; H, 8.30; N, 2.32.
LSIMS (negative-ion mode, 2:2:1 diethanolamine/tet-
ramethlyurea/m-nitrobenzyl alcohol as the liquid ma-
trix): 1648 [M−H]−, 1399 [M−H−Neu]−, 1237
[1399−Gal]−, 888 [lactosyl ceramide]−, 726 [glucosyl
ceramide]−.
Selectin binding assays.—Binding of the recombinant
E-, L- and P-selectins (Fcm chimeras)15 to glycolipids
immobilized in microwells was assayed as described
previously16 after harvesting the glycolipids by prepara-
tive TLC.7 Binding to the target molecule, N-deacetyl
sLex, was compared with binding to the gangliosides
sLex (GSC 64)17 6-O-sulfo-sLex (GSC 269)6 and 6-O-
sulfo-N-deacetyl sLex (GSC 406).9 Galactosyl ceramide
(Sigma) was included as a negative control.
D
L
D
D
D
1)-(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-octa-
decene-1,3-diol (14).—H2S was bubbled through a
stirred solution of 13 (71 mg, 28 mmol) in pyridine (12.4
mL) and water (2.5 mL) for 72 h at 0 °C. The mixture
was concentrated, and the residual syrup was treated
with octadecanoic acid (24 mg, 84 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
hydro-
genchloride (WSC; 23 mg, 0.12 mmol) in CH2Cl2 (3
mL) 12 h at rt. The mixture was extracted with CHCl3
and the extract was successively washed with 1 M HCl
and water, dried (Na2SO4) and concentrated. Column
chromatography (50:1 CHCl3–MeOH) of the residue
on silica gel gave 13 (33 mg, 43%) as an amorphous
mass: [h]D −13.1° (c 6.6, CHCl3). 1H NMR (CDCl3): l
0.89 (t, 6 H, JMe,CH2 6.9 Hz, 2 MeCH2), 1.27 (s, 52 H,
26 CH2), 1.57 (s, 3 H, AcN), 1.67 (dd, 1 H, Jgem 12.5,
J3ax,4 12.3 Hz, H-3Vax), 1.84–2.14 (14 s, 42 H, 14 AcO),
2.50 (dd, 1 H, J3eq,4 4.6 Hz, H-3Veq), 3.84 (s, 3 H,
COOMe), 4.40 (d, 1 H, J1,2 7.7 Hz, H-1I), 4.70 (dd, J1,2
2.9, J2,3 7.3 Hz, H-2VI), 4.94 (d, 1 H, J1,2 7.3 Hz, H-1IV),
5.07 (d, 1 H, H-1VI), 5.26 (dd, 1 H, J6,7 3.8, J7,8 9.8 Hz,
H-7V), 5.45 (dd, 1 H, J2,3 7.7 Hz, H-2IV), 5.66 (m, 1 H,
H-8V), 5.88 (dt, 1 H, J4,5 14.7, J5,6=J5%,6 7.3 Hz, H-5 of
sphingosine), 6.25 (d, 1 H, J5,NH 8.6 Hz, NHV), 7.45–
8.18 (m, 20 H, 4 Ph). Anal. Calcd for C136H184F3N3O53
(2765.93): C, 59.06; H, 6.71; N, 1.52. Found: C, 59.02;
H, 6.51; N, 1.48.
Acknowledgements
This study was supported in part by Grants-in-Aid
(No. 12306007) for Scientific Research from the Min-
istry of Education, Science, and Culture of Japan, and
Japan Society for Promotion of Science. We are grate-
ful to Dr. Wengang Chai for the mass spectrometric
analyses.
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