Synthesis of sialyl Lewis X pentasaccharide analogue for high-throughput screening of selectin blockers

Article abstract of DOI:10.1016/S0968-0896(98)00003-0

We have developed an effective synthesis of sLe(x) pentasaccharide glycolipid analogue 2. As a part of application of sLe(x) pentasaccharide glycolipid 2 synthesized here, we have investigated the construction of a high-throughput screening system for discovery of selectin blockers. As a result, it was found that compound 2 was a useful ligand for in vitro ELISA assay and could be an important material for high-throughput screening of selectin blockers. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00003-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 587±593
Synthesis of Sialyl Lewis X Pentasaccharide Analogue for
High-throughput Screening of Selectin Blockers
Takao Kiyoi,a Yoshimasa Inoue,b Hiroshi Ohmoto,b Masahiro Yoshida,a
Makoto Kisoc and Hirosato Kondoa,*
aDepartmentofMedicinalChemistry,Kanebo,NewDrugResearchLaboratories,1-5-90Tomobuchi-Cho,Miyakojima-Ku,Osaka534,Japan
bDepartment of Molecular Biology, Kanebo, New Drug Research Laboratories, 1-5-90 Tomobuchi-Cho, Miyakojima-Ku, Osaka 534, Japan
cDepartment of Applied Bioorganic Chemistry, Gifu University, Gifu 501-11, Japan
Received 23 October 1997; accepted 22 December 1997
AbstractÐWe have developed an e€ective synthesis of sLex pentasaccharide glycolipid analogue 2. As a part of appli-
cation of sLex pentasaccharide glycolipid 2 synthesized here, we have investigated the construction of a high-through-
put screening system for discovery of selectin blockers. As a result, it was found that compound 2 was a useful ligand
for in vitro ELISA assay and could be an important material for high-throughput screening of selectin blockers.
# 1998 Elsevier Science Ltd. All rights reserved.
Introduction
throughput screening of selectin blockers, and using this
ELISA assay system, we have already discovered some
potent selectin blockers in vitro and in vivo.11 For the
e€ective research of selectin blockers, one of the most
important things is to construct in vitro ELISA assay
system which involves an inhibitory activities of selectin
blockers toward sLex-selectin binding. However, our
ELISA assay system reported previously10 has some
points to improve, because a synthetic sLex penta-
saccharide linked to natural ceramide (1) necessary for
ELISA assay is dicult and expensive to produce, at
least for now. Especially, the sLex pentasaccharide and
natural ceramide require several complicated and
expensive synthesis steps. Therefore, to establish more
practical in vitro ELISA assay system we should over-
come the issue, how easy to make sLex pentasaccharide
analog. For a start, we focused on the synthesis of
simpler sLex glycolipid analog. Namely, we selected a
3-tridecylhexadecyl group as an alternative to a cer-
amide. The chain length of this group seems to be slight
shorter than the natural ceramide, and it has not yet
been reported whether the natural ceramide of sLex
glycolipid could be replaced by a simple alkyl chain such
as 3-tridecylhexadecyl group.
Selectins are a family of cell adhesion molecules which
participate in the initial phase of adhesion between leu-
kocytes and endothelial cells, and play an important
role in leukocyte in®ltration into tissue in in¯ammatory
reactions.1 Recent studies have indicated that the ligand
recognized by selectins was a carbohydrate,2 e.g. sialyl
Lewis X (sLex), and a selectin-carbohydrate interaction
occurred at the very early stage of in¯ammatory reac-
tion called `rolling'.3 Selectins are expressed on a variety
of cell surfaces. For example, E-selectin is expressed on
vascular endothelial cells during in¯ammation.4 P-selec-
tin is expressed on platelets and vascular endothelial
cells,5 and L-selectin is an adhesion molecule expressed
on leukocytes.6 The role of selectins in diseases has been
gradually evident through the several experiments using
mAb7 and/or knockout mice8 of selectins. In addition,
recently it has been reported that selectins played an
important role in angiogenesis and a selectin blocker
inhibited angiogenesis in vitro and in vivo.9 Such a
integral event in vivo makes the selectins attractive targets
for the therapy of these diseases.
In a series of study of selectin blockers, we have recently
reported a useful in vitro ELISA assay10 for high-
By the way, the 3-tridecylhexadecyl group is com-
mercially available and would be quite readily intro-
duced to the anomeric position of sLex.12 Therefore, the
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00003-0

588
T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
replacement of natural ceramide to simpler chain such
as 3-tridecylhexadecyl group would remarkably support
the ecient ELISA assay system, especially for the
high throughput screening. Our goal is an e€ective
construction method of sLex pentasaccharide and a new
®nding of ceramide analogue instead of the natural cer-
amide.
donor 1013 to provide the sLex pentasaccharide 11 in an
82% yield.
We next investigated the incorporation of a branched
alkyl chain to the sLex pentasaccharide skeleton. As
shown in Scheme 2, the hydrogenolysis of the benzyl
groups of 11 in the presence of a catalytic amount of
10% Pd-C, followed by the acetylation of free hydroxyl
groups in the presence of acetic anhydride and pyridine
a€orded a compound 12 in a moderate yield. The
removal of 2-(trimethylsilyl)ethyl (SE) group of 12 with
tri¯uoroacetic acid was followed by the treatment with
trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]lundec-
7-ene (DBU) in chloroform at 0 ꢀC to a€ord the glycosyl
donor 13 in a good yield. The glycosylation of 3-tridecyl-
hexadecanol 1412 with the trichloroacetimidate donor
13, in dichloromethane in the presence of boron tri-
¯uoride etherate, gave exclusively the b-glycoside 15, in
a 74% yield, because of the neighboring e€ect of the C-2
acetyl group of donor 13. Finally, the compound 15 was
transformed quantitatively, by the removal of the pro-
tecting groups, into the desired compound 2.
In this paper, we describe the e€ective synthesis of 3-
tridecylhexadecyl-sLex pentasaccharide 2, which would
be easier to make than sLex pentasaccharide linked to
natural ceramide 1, and the comparison of the ligand
avidity of compounds 1 and 2 using the known selectin
blocker.
Results and Discussion
Chemistry
We have already reported13 the synthesis of sLex penta-
saccharide 3, which involves the quantitative construc-
tion of a tetrasaccharide, NeuAca(2!3) Galb(1!4)
GlcNAcb(1!3) GalbOEt, and an e€ective incorpora-
tion of fucose moiety toward the tetrasaccharide. As a
part of the application of this synthetic method, we
investigated a synthesis of sLex pentasaccharide glycoli-
pid analogue 2, which would be a useful material for in
vitro ELISA assay.
Biological activities: Investigation of in vitro ELISA
assay using 3-tridecylhexadecyl-sLex pentasaccharide 2
According to our previous paper,10 we established the
ELISA inhibition assay system (Figure 1; Schematic dia-
gram) using the selectin-Ig chimera and 3-tridecylhexa-
decyl- sLex pentasaccharide 2, and evaluated the
known selectin blocker 4 (Figure 2). As shown in Table 1,
compound 4 gave the similar IC50 values toward E-
selectin/ ligands 1 and/or E-selectin/ligand 2 bindings
(14 mM for ligand 1 and 16 mM for ligand 2). In addi-
tion, against the P- and L-selectins, each sLex penta-
saccharide 1 and 2 gave the quite similar IC50 values
toward 4, 46 mM, 49 mM for P-selectin and 22 mM,
21 mM for L-selectin, respectively. As shown in Table 1,
both compounds 1 and 2 had very closed ligand avidity
toward E-, P-, and L-selectins binding in spite of having
a di€erent chains, natural ceramide and arti®cial bran-
ched alkyl chain, respectively. Namely, although the
chain length of 3-tridecylhexadecyl group is a little bit
shorter than that of natural ceramide, it was proved
that 3-tridecylhexadecyl group was enough length for
coating on the well and did not a€ect the selectin
binding.
As shown in Scheme 1, GlcNAcb(1!3)GalbOSE
acceptor 6 was obtained from disaccharide 514 by the
combination of protection and/or deprotection of the
hydroxyl groups according to the established methods,
i.e. 4-methoxybenzylation of the free hydroxyl group
and selective cleavage of 40,60-O-benzylidene moiety.
The glycosylation of 6 with the NeuAca(2!3)GalbSMe
donor 713 in the presence of dimethyl(methylthio) sul-
fonium tri¯ate15 (DMTST) as a promoter gave the
tetrasaccharide 8 in 77% yield. Removal of the 4-meth-
oxybenzyl (MPM) group of 8 gave the tetrasaccharide 9
quantitatively, which was glycosylated with a fucose
In conclusion, we succeeded in an e€ective synthesis of
sLex glycolipid analogue 2. In addition, we have con-
structed an ELISA assay system using the sLex glyco-
lipid 2 and evaluated inhibitory activities of the known
selectin blocker. As a result, it was found that com-
pound 2 could also be a useful ligand for the in vitro
ELISA assay and our ELISA assay system constructed
here was a convenient evaluation system for discovery
Chart 1.

T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
589
Scheme 1. Reagents and conditions: (i) NaH, MPMCl, DMF (100%); (ii) NaBH3CN/HCl (53%); (iii) DMTST, CH2Cl2 (77%); (iv)
Ce(NH4)2(NO3)6, CH3CN (100%); (v) NlS/TfOH, CH2Cl2 (82%).
Scheme 2. Reagents: (a) H2/10%Pd/C, Ac2O/pyridine, DMAP (75%); (b) (i) CF3CO2H, CHCl3 (93%), (ii) CCl3CN/DBU, CHCl3
(85%); (c) 3-tridecylhexadecanol (14), BF3/OEt2, CH2Cl2 (74%); (d) NaOMe, MeOH (100%).

590
T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
Figure 1. Schematic diagram of ELISA inhibition assay using selectin-Ig chimera. For details see Experimental.
of selectin blockers. These ®ndings indicate that sLex
glycolipid 2 could be an important material for high-
throughput screening of selectin blockers.
in a 1:1 mixture of methanol and distilled water, was
pipetted into microtiter plate wells (96 wells, Falcon
PRO-BIND) at 100 pmol/50 mL/well, and was adsorbed
by evaporating the solvent. The wells were blocked with
5% BSA (bovine serum albumin) 1 mM CaCl2/50 mM
imidazole bu€er (pH 7.2) for 1 h at room temperature,
and washed three times with 50 mM imidazole bu€er
(pH 7.2).
Experimental
In vitro ELISA assay of E-, P-, and L-selectin - sLex
binding
Separately, the following were added to 1% BSA 1 mM
CaCl2/50 mM imidazole bu€er (pH 7.2): (a) 1:500
dilution of biotinylated goat F(ab0)2 anti-human IgG(g)
(BIOSOURCE); (b) 1:500 dilution of streptavidin-
alkaline phosphatase (Zymed Lab Inc.); and (c) 10 mg/
ml E-selectin IgG chimera, 20 mg/ml P-selectin IgG chi-
mera, or 5 mg/ml L-selectin IgG chimera to form a
complex. The test compound (4) was dissolved in dis-
tilled water at 10 mM and diluted by 1% BSA 1 mM
CaCl2/50 mM imidazole bu€er (pH 7.2) to ®nal con-
centrations at 200, 100, 50, 25, 12.5, 6.25, 3.13, and
1.56 mM, respectively. Reactant solutions were prepared
by incubating 30 mL of this solution at each concentra-
tion with 30 mL of the above complex solution for
30 min at room temperature. This reactant solution was
then added to the above microtiter wells at 50 mL/well
and incubated at 37 ꢀC for 45 min. The wells were
washed three times with 50 mM imidazole bu€er (pH
7.2) and distilled water respectively, followed by addi-
tion of p-nitrophenylphosphate (1 mg/mL) and 0.01%
of MgCl2 in 1 M diethanolamine (pH 9.8) at 50 mL/well.
The reactant mixture was developed for 120 min at
room temperature and absorbance at 405 nm was
The construction of the selectin-immunoglobulin was
carried out according to the previous paper.2f
A solution of sLex pentasaccharide ceramide 1 or 1-(3-
tridecyl)hexadecyl sLex pentasaccharide 2, respectively,
Figure 2. Selectin blockers (3,4).

T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
591
Table 1. Blocking activity of compound 4 using the glycolipids
1 and 2
ane:ethyl acetate, gradient elution to 1:2) of the residue
on silica gel gave 6 (561 mg, 53%): 1H NMR(CDC13,
250 MHz) d; 0.01 (s, 9H, SiMe3), 0.95±1.05 (m, 2H,
CH2Si), 1.55 (s, 3H, NHAc), 3.80 (s, 3H, OMe), 4.79 (d,
J=9 Hz, 1H, CH2Ph), 4.85 (d, J=8 Hz, 1 H, H-1,
GlcNAc), 4.90 (d, J=12 Hz, 1 H, CH2Ph), 5.06 (d,
J=12 Hz, 2H, CH2Ph), 6.86 (d, J=9 Hz, 2H, MPM),
7.21 (d, J=9 Hz, 2H, MPM), 7.25±7.45 (m, 20H, aro-
matic H). M.S. (m/e) (M+Na)+: 986.
IC50, mM
E-selectin P-selectin L-selectin
Compd/glycolipids
4
1
14
2
16
1
46
2
49
1
22
2
21
measured. Percent binding was calculated by the fol-
lowing equation:
2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-
O - acetyl - 3,5 - dideoxy - D - glycero - ꢁ - D - galacto - 2-
nonulopyranosylonate)-(2!3)-O-(2,4,6-tri-O-benzoyl-ꢀ-
D-galactopyranosyl)-(1!4)-O-[2-acetamido-6-O-benzyl-
2-deoxy-3-O-(4-metboxybenzyl)-ꢀ-D-glucopyranosyl]-
(1!3)-2,4,6-tri-O-benzyl-ꢀ-D-galactopyranoside (8). The
compound 6 (150 mg, 0.156 mmol) and methyl O-(methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosylonate)-(2!3)-2,4,6-tri-
O-benzoyl-1-thio-b-D-galactopyranoside (7) (201 mg,
0.202 mmol) were dissolved in dry CH2C12 (3.0 mL) and
stirred over 4 A molecular sieves (powder, 400 mg) at
room temperature for 15 h. The mixture was then cooled
to 0 ꢀC, and the mixture of DMTST (281 mg, 1.089 mmol)
and 4 A molecular sieves (powder, 212 mg) was added.
The mixture was stirred for 24 h at 7 ꢀC. To the reaction
mixture, methanol (1.0 mL), triethylamine (1.0 mL) and
ethyl acetate (10 mL) were added, then the mixture was
®ltered and washed with water. The aqueous layer was
extracted with ethyl acetate and the organic layer was
washed with brine, and dried over anhydrous MgSO4,
then the solvent was removed in vacuo. The residue
was subjected to chromatography (5:1 toluene:acetone,
gradient elution to 4:1) to a€ord 8 (229 mg, 77% yield)
% Binding ˆ ꢀX C=A C†  100
wherein X is the absorbance of wells containing the test
compound at each concentration, C is the absorbance of
wells not containing the selectin-Ig and test compound,
and A is the absorbance of control wells not containing
the test compound. The results of inhibitory activities
are presented in Table 1 as IC50 values. The number of
replicates is two.
2-(Trimethylsilyl)ethyl-O-[2-acetamido-6-O-benzyl-2-
deoxy - 3 - O - (4 - methoxybenzyl) - ꢀ - D - glucopyranosyl]-
(1!3)-2,4,6-tri-O-benzyl-ꢀ-D-galactopyranoside (6). To
a solution of 2-(trimethylsilyl)ethyl O-(2-acetamido-4,6-
O-benzylidene-2-deoxy-b-D-glucopyranosyl)-(1!3)-2,4,6-
tri-O-benzyl-b-D-galactopyranoside(5)14 (925 mg, 1.10
mmol) in DMF (4.0 mL) was added a suspension of
sodium hydride in oil (44 mg, 60% of sodium hydride by
weight). The mixture was stirred for 20 min at 0 ꢀC,
4-methoxybenzyl chloride (194 mL, 1.43 mmol) was add
dropwise, and stirring was continued for 4 h at room
temperature. Moreover, a suspension of sodium hydride
in oil (22 mg, 60% of sodium hydride by weight) was
add to the reaction mixture, and stirring was continued
for 6 h at room temperature. The reaction mixture was
cooled to 0 ꢀC, and methanol (2 mL) and ethyl acetate
(20 mL) were added, then the mixture was washed with
water, and extracted with ethyl acetate. The extract was
washed with brine, dried (Na2SO4), and concentrated.
Column chromatography (chloroform) of the residue on
silica gel gave 2-(trimethylsilyl)ethyl O-[2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-(4-methoxybenzyl)-b-D-
glucopyranosyl]-(1!3)-2,4,6-tri-O-benzyl-b-D-galacto-
pyranoside (1.07 g, 100%). To a solution of the latter
compound (1.07 g, 1.10 mmol) in THF (15 mL) was
added 4 A molecular sieves (powder, 1.0 g), and the
mixture was stirred for 2 h at room temperature, and
sodium cyanoborohydride (992 mg) was added portion-
wise. After the reagent had dissolved, 1.0 M hydrogen
chloride in ether was added dropwise at 0 ꢀC until the
evolution of gas ceased. The mixture was neutralized
with triethylamine and ®ltered, the residue was washed
with methanol and the combined ®ltrate and washings
were concentrated. Column chromatography (2:1 hex-
1
as a white solid: H NMR(CDC13, 250 MHz) d; 0.02
(s, 9H, SiMe3), 0.97 (t, J=9 Hz, 2H, CH2Si), 1.54 (s,
3H, NHAc), 1.56 (s, 3H, NHAc), 1.67 (t, J=12 Hz, 1H,
Neu-3a), 1.79 (s, 3H, OAc), 1.92 (s, 3H, OAc), 1.98 (s,
3H, OAc), 2.15 (s, 3H, OAc), 2.46 (dd, J=5, 13 Hz, 1H,
Neu-3e), 3.67 (s, 3H, CO2Me), 3.83 (s, 3H, OMe), 5.08
(d, J=8 Hz, 1H, GlcNAc H-1), 5.23 (dd, J=3, 10 Hz,
1H, Neu, H-7), 5.60±5.73 (m, 1H, Neu, H-8), 7.10±8.30
(m, 35H, aromatic H). M.S. (m/e) (M+Na)+: 1935.
2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-
O - acetyl - 3,5 - dideoxy - D - glycero - ꢁ - D - galacto - 2-
nonulopyranosylonate)-(2!3)-O-(2,4,6-tri-O-benzoyl-ꢀ-
D-galactopyranosyl)-(1!4)-O-(2-acetamido-6-O-benzy]-
2-deoxy-ꢀ-D-glucopyranosyl)-(1!3)-2,4,6-tri-O-benzyl-
ꢀ-D-galactopyranoside (9). To a solution of 8 (407 mg,
0.213 mmol) in a mixture of acetonitrile (4.0 mL) and
water (0.5 mL) was added cerium (IV) diammonium
nitrate (282 mg, 0.516 mmol). The solution was stirred at
room temperature for 4 h, then to the mixture ethyl
acetate (20 mL) was added and washed with water. The
aqueous layer was extracted with ethyl acetate and the

592
T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
combined organic layer was washed with saturated
aqueous NaHCO3, water, and brine, and dried over
MgSO4, then the solvent was removed in vacuo. Col-
umn chromatography (1:1 hexane:ethyl acetate, gra-
dient elution to 1:4) of the residue on silica gel gave 9
nonulopyranosylonate)-(2!3)-O-(2,4,6-tri-O-benzoyl-ꢀ-
D-galactopyranosyl)-(1!4)-O-[(2,3,4-tri-O-acetyl-ꢁ-L-
fucopyranosyl)-(1!3)]-O-(2-acetamido-6-O-acetyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1!3)-2,4,6-tri-O-acetyl-ꢀ-
D-galactopyranoside (12). A solution of 11 (370 mg,
0.168 mmol) in ethanol (30 mL) and acetic acid (15 mL)
was hydrogenolyzed in the presence of 10 % Pd-C
(400 mg) for 20 h at 50 ꢀC, then ®ltered and con-
centrated. The residue was acetylated with acetic anhy-
dride (4.0 mL)-pyridine (8.0 mL) for 3 h in the presence
of DMAP (2.0 mg) at 40 ꢀC. The product was puri®ed
by chromatography (1: 2 hexane : ethyl acetate, gradient
elution to ethyl acetate) on silica gel to give 12 (236 mg,
75%): 1H NMR(CDCl3, 250 MHz) d; 0.01 (s, 9H,
SiMe3), 0.85±0.95 (m, 2H, CH2Si), 1.20 (d, J=7 Hz, 3H,
Fuc H-6), 1.56 (s, 3H, NHAc), 1.90 (s, 3H, 0Ac), 1.93 (s,
3H, OAc), 2.00 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.03 (s,
3H, OAc), 2.04 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.06 (s,
3H, OAc), 2.07 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.09 (s,
3H, OAc), 2.12 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.41
(dd, J=4, 12 Hz, 1 H, Neu-3e), 3.81 (s, 3H, CO2Me),
5.30±5.45 (m 1 H, Neu , H-7), 5.55±5.70 (m, 1 H, Neu,
H-8), 7.25±8.30 (m, 15H, aromatic H).
1
(385 mg, 1 00%): H NMR(CDCl3, 250 MHz) d; 0.03
(s, 9H, SiMe3) 0.92±1.00 (m, 2H, CH2Si), 1.55 (s, 3H,
NHAc), 1.58 (s, 3H, NHAc), 1.63 (t, J=12 Hz, 1H,
Neu-3a), 1.79 (s, 3H, OAc), 1.91 (s, 3H, 0Ac), 2.03 (s,
3H, OAc), 2.19 (s, 3H, OAc), 2.46 (dd, J=5, 13 Hz, 1H,
Neu-3e), 3.87 (s, 3H, CO2Me), 5.00 (d, J=10 Hz, 1H,
GlcNAc H- 1), 5.22 (dd, J=3, 10 Hz, 1H, Neu, H-7),
5.60±5.72 (m 1H, Neu, H-8), 7.00±8.20 (m, 35H, aro-
matic H). M.S. (m/e) (M+Na)+: 1813.
2-(Trimethylsilyl)ethyl-O-(methyl 5-acetamido-4,7,8,9-tetra-
O - acetyl - 3,5 - dideoxy - D - glycero - ꢁ - D - galacto - 2-
nonulopyranosylonate)-(2!3)-O-(2,4,6-tri-O-benzoyl-ꢀ-
D-galactopyranosyl)-(1!4)-O-[(2,3,4-tri-O-benzyl-ꢁ-L-
fucopyranosyl)-(1!3)]-O-(2-acetamido-6-O-benzyl-2-
deoxy-ꢀ-D-glucopyranosyl)-(1!3)-2,4,6-tri-O-benzyl-ꢀ-
D-galactopyranoside (11). To a solution of 9 (403 mg,
0.225 mmol) and phenyl 2,3,4-tri-O-benzyl-1-thio-b-L-
fucopyranoside (10) (237 mg, 0.450 mmol) in toluene
(4.0 mL) and dichloromethane (2.0 mL), was added 4 A
molecular sieves (powder, 500 mg), and the mixture was
stirred for 13 h at room temperature. N-Iodosuccinimide
(151 mg, 0.675 mmol) and tri¯uoromethanesulfonic acid
(3 mL, 0.034 mmol) were added to the mixture at 20 ꢀC,
and the stirring was continued for 4 h at 15 ꢀC. More-
over, 10 (237 mg), N-iodosuccinirnide (151 mg), and tri-
¯uoromethanesulfonic acid (3 mL) were added to the
mixture at 15 ꢀC, and the mixture was stirred for 3 h at
15 ꢀC. Ethyl acetate was added to the mixture, the
insoluble materials were ®ltered o€, and the ®ltrate was
washed with saturated aqueous NaHCO3. The aqueous
layer was extracted with ethyl acetate and the organic
layer were combined, washed with saturated sodium
thiosulfate solution, water, and brine, and dried
(MgSO4). Column chromatography (2:1 hexane : ethyl
acetate, gradient elution to 1:4) on silica gel gave 11
(406 mg, 82%): 1H NMR(CDCl3, 25O MHz) d; 0.01
(s, 9H, SiMe3), 0.98 (t, J=9 Hz, 2H, CH2Si), 1.14
(d, J=6 Hz, 3H, Fuc H-6), 1.58 (s, 3H, NHAc), 1.61 (s,
3H, NHAc), 1.70 (t, J=12 Hz, 1H, Neu-3a), 1.80 (s, 3H,
OAc), 1.92 (s, 3H, OAc), 1.97 (s, 3H, OAc), 2.15 (s, 3H,
OAc), 2.43 (dd, J=4, 12 Hz, 1H, Neu-3e), 3.84 (s, 3H,
CO2Me), 4.57 (d, J=13 Hz, 1H, CH2Ph), 4.84
(d, J=12 Hz, 1H, CH2Ph), 5.00 (d, J=8 Hz, 1H,
GlcNAc, H-1), 5.26 (d, J=2 Hz, 1H, Fuc, H-1), 5.40±
5.50 (m, 1 H, Neu , H-7), 5.60±5.75 (m, 1 H, Neu,
H-8), 7.10±8.30 (M 50H, aromatic H). M.S. (m/e)
(M+Na)+: 2230.
O-(Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D-glycero-ꢁ-D-galacto-2- nonulopyranosylonate)-(2!3)-
O-(2,4,6-tri-O-benzoyl-ꢀ-D-galactopyranosyl)-(1!4)-O-
[(2,3,4-tri-O-acetyl-ꢁ-L-fucopyranosyl)-(1!3)]-O-(2-
acetamido-6-O-acetyl-2-deoxy-ꢀ-D-glucopyranosyl)-(1!3)-
2,4,6-tri-O-acetyl-ꢁ-D-galactopyranosy1 trichloroacet-
imidate (13). To a solution of 12 (96 mg, 0.51 mmol) in
chloroform (0.8 mL) was added tri¯uoroacetic acid
(0.6 mL) at 0 ꢀC, and the mixture was stirred for 90 min
at room temperature and concentrated. Column chro-
matography (1:4; hexane : ethyl acetate, gradient elution
to ethyl acetate) on silica gel gave O-(methyl 5-acet-
amido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosylonate)-(2!3)-O-(2,4,6-tri-O-
benzoyl-b-D-galactopyranosyl)-(1!4)-O-[(2,3,4-tri-O-
acetyl-a-L-fucopyranosyl)-(1!3)]-O-(2-acetamido-6-O-
acetyl-2-deoxy-b-D-glucopyranosyl)-(1!3)-2,4,6-tri-O-
acetyl-D-galactopyranose (85 mg, 93%). To a mixture of
this pentasaccharide (58 mg, 0.033 mmol) and tri-
chloroacetonitrile (0.1 mL) in chloroform (1.0 mL)
was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU,
5.2 mg) at 0 ꢀC, and the mixture was stirred for 100 min
at the same temperature, then concentrated. Column
chromatography (1:2; hexane:ethyl acetate, gradient
elution to ethyl acetate) on silica gel a€orded 13 (53 mg,
1
85%). H NMR(CDCl3, 250 MHz) d; 1.21 (d, J=7 Hz,
3H, Fuc- 6), 1.58 (s, 3H, NAc), 1.78 (s, 3H, NAc), 1.85-
2.15 (11s, 33H, OAc), 2.41 (dd, J=5, 13 Hz, 1H, Neu-
3e), 3.81 (s, 3H, MeO), 5.55±5.70 (m, 1H, Neu-8),
6.50 (d, J=4 Hz, 1H, Gal-1), 7.40±8.20 (m 15H,
aromatic H), 8.61 (s, 1H, C=NH). M.S. (m/e)
(M+Na)+: 1938.
2-(Trimethylsilyl)ethyl O-(methyl 5-acetamido-4,7,8,9-tetra-
O - acetyl - 3,5-dideoxy - D - glycero - ꢁ - D - galacto - 2-

T. Kiyoi et al./Bioorg. Med. Chem. 6 (1998) 587±593
593
3-Tridecylhexadecyl O-(methyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-D-glycero-ꢁ-D-galacto-2-nonulopyr-
anosylonate)-(2!3)-O-(2,4,6-tri-O-benzoyl-ꢀ-D-galacto-
pyranosyl)-(1!4)-O-[(2,3,4-tri-O-acetyl-ꢁ-L-fucopyrano-
syl)-(1!3)]-O-(2-acetamido-6-O-acetyl-2-deoxy-ꢀ-D-
glucopyranosyl)-(1!3)-2,4,6-tri-O-acetyl-ꢀ-D-galactopyr-
anoside (15). To a solution of 13 (25 mg, 0.013 mmol)
and 3-tridecylhexadecanol (14, 28 mg) in dichloro-
methane (0.3 mL) was added 4 A molecular sieves type
AW300 (powder, 56 mg), and the mixture was stirred
for 4 h at room temperature, then cooled to 0 ꢀC. Boron
tri¯uoride etherate (8 mg) was added, and the mixture
was stirred for 18 h at 0 ꢀC and then ®ltered. The inso-
luble material was washed with ethyl acetate, and the
combined ®ltrate and washings were washed with satu-
rated aqueous NaHCO3 and water, dried (Na2SO4) and
concentrated. Column chromatography (hexane: ethyl
acetate 1: 1, gradient elution to ethyl acetate) on silica
gel gave 15 (21 mg, 74%): [a]D 10ꢀ (c 0.59, CHCl3); 1H
NMR(CDCl3, 25O MHz) d; 0.89 (t, J=7 Hz, 6H, CH3),
1.15±1.40 (m 48H), 1.21 (d, J=7 Hz, 3H, Fuc-6), 1.78 (s,
3H, NAc), 1.82 (s, 3H, NAc), 1.90 (s, 3H, OAc), 1.93 (s,
3H, OAc), 2.00 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.05 (s,
3H, OAc), 2.07 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.09 (s,
3H, OAc), 2.12 (s, 3H, OAc), 2.41 (dd, J=4, 13 Hz, 1H,
Neu-3e), 3.81 (s, 3H, COOCH3), 5.60±5.70 (m, 1H,
Neu-8), 7.40±8.20 (m, 15H, aromatic H). M.S. (m/e)
(M+Na)+: 1793.
Cell 1991, 65, 859. (c) Watson, S. R.; Fennie, C.; Laskey, L. A.
Nature 1991, 349, 164. (d) Mayades, T. N.; Johnson, R. C.;
Rayburn, H.; Hynes, R. O.; Wagner, D. D. Cell 1993, 74, 541.
2. (a) Phillips, M. L.; Nudelman, E.; Gaeta, F. C. A.; Perez, M.;
Singhal, A. K.; Hakomori, S.-T.; Paulson, J. C. Science 1990, 250,
1130. (b) Walz, G.; Aru€o, A.; Kolanus, W.; Bevilacqua, M.;
Seed, B. Science 1990, 250, 1132±1134. (c) Watson, S. A.; Imai,
Y.; Fennie, C; Geo€roy, J. S.; Rosen, S. D.; Lasky, L. A. A. J.
Cell Biol. 1990, 110, 2221. (d) Aru€o, A.; Kolanus, W.; Walz,
G.; Fredman, P.; Seed, B. Cell 1991, 67, 35. (e) Erbe, D. V.;
Watson, S. R.; Presta, L. G.; Wolitzky, B. A.; Foxall, C.;
Brandley, B. K.; Lasky, L. A. J. Cell. Biol. 1993, 120(5), 1227.
(f) Faxall, C.; Watson, S. R.; Dowbenko, D.; Fennie, C.;
Lasky, L. A.; Kiso, M.; Hasegawa, A.; Brandley, B. K. J. Cell
Biol. 1992, 117, 895.
3. Tedder, T.; Steeber, D.; Chen, A.; Engel, P. FASEB J. 1995,
9, 866.
4. Bevilacqua, M. P.; Stengelin, S.; Gimbrone, M. A. Science
1989, 243, 1160.
5. Isenberg, W. M.; MeEver, R. P.; Shuman, M. A.; Bainton,
D. F. Blood Cells 1986, 12, 191.
6. Butcher, E. C. Cell 1991, 67, 1033.
7. (a) Mulligan, M. S.; Polley, M. J.; Bayer, R. J.; Nunn, M.
F.; Paulson, L. C.; Ward, P. A. J. Clin. Invest. 1992, 90, 1600.
(b) Weyrich, A. S.; Ma, X.; Lefer, D. J.; Albertine, K. H.;
Lefer, A. M. J. Clin. Invest. 1993, 91, 2620. (c) Winn, R. K.;
Liggitt, D.; Vedder, N. B.; Paulson, L. C.; Harlan, J. M. J.
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1994, 28, 1414.
3 - Tridecylhexadecyl O - (5 - acetamido - 3,5 - dideoxy - D-
glycero-ꢁ-D-galacto-2-nonulopyranosylonic acid)-(2!3)-
O-(ꢀ-D-galactopyranosyl)-(1!4)-O-[(ꢁ-L-fucopyranosyl)-
(1!3)]-O-(2-acetamido-2-deoxy-ꢀ-D-glucopyranosyl)-
(1!3)-ꢀ-D-galactopyranoside (2). To a solution of 15
(46 mg, 0.021 mmol) in methanol (10 mL) was added
sodium methoxide (8 mg), and the mixture was stirred
for 14 h at 50 ꢀC, and water (1.0 mL) was added. The
solution was stirred for 24 h at 50 ꢀC, then treated with
DOWEX (5OW-X8, H+ form) resin and ®ltered. The
resin was washed with methanol, and combined ®ltrate
and washings were concentrated. Column chromato-
graphy (5:4 chloroform: methanol) of the residue on
Sephadex LH-20 gave 2 (29 mg, 100%) as an amor-
8. Frenette, P. S.; Wagner, D. D. Thrombosis and Haemostasis
1997, 78, 60.
9. Nguyen, M.; Eilber, F. R.; Defrees, S. Biochem. Biophys.
Res. Commun. 1996, 228, 716.
10. Ohmoto, H.; Nakamura, K.; Inoue, T.; Kondo, N.; Inoue,
Y.; Yoshino, K.; Kondo, H.; Ishida, H.; Kiso, M.; Hasegawa,
A. J. Med. Chem. 1996, 39, 1339.
11. (a) Wada, Y.; Saito, T.; Matsuda, N.; Ohmoto, H.; Yosh-
ino, K.; Ohashi, M.; Kondo, H.; Ishida, H.; Kiso, M.; Hase-
gawa, A. J. Med. Chem. 1996, 39, 2055. (b) Tsukida, T.;
Yoshida, M.; Kurokawa, K.; Nakai, Y.; Achiha, T.; Kiyoi, T.;
Kondo, H. J. Org. Chem. 1997, 62, 6976. (c) Tsukida, T.; Hir-
amatsu, Y.; Tsujishita, H.; Kiyoi, T.; Yoshida, M.; Kurokawa,
K.; Modyama, S.; Ohmoto, H.; Wada, Y.; Saito, T.; Kondo,
H. J. Med. Chem. 1997, 40, 3534.
phous mass: [a]D 15ꢀ (c 0.49,1 : 1; CHCl3-MeOH); H
1
NMR(CD3OD, 250 MHz) d; 0.90 (t, J=6 Hz, 6H, CH3),
1.17 (d, J=7 Hz, 3H, Fuc-6), 1.20±1.40 (m, 48H), 1.89
(t, J=12 Hz, 1H, Neu-3a), 1.98 (s, 3H, NAc), 2.00 (s,
3H, NAc), 2.78 (dd, J=4, 13 Hz, 1H, Neu-3e), 4.22 (d,
J=7 Hz, 1H,), 4.52 (d, J=8 Hz, 1H,), 5.06 (d, J=4 Hz,
1H, Fuc- 1). M.S. (m/e) (M+Na)+: 1413.
12. Hasegawa, A.; Suzuki, N.; Ishida, H.; Kiso, M.; J. Carb-
hydr. Chem. 1996, 15, 623.
13. Kiyoi, T.; Nakai, Y.; Kondo, H.; Ishida, H.; Kiso, M.;
Hasegawa, A. Bioorg. Med. & Chem. 1996, 4, 1167.
14. Hasegawa, A.; Ando, T.; Kameyama, A.; Kiso, M. J.
Carbohydr. Chem. 1992, 11, 645.
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