Synthesis and glycan priming activity of acetylated disaccharides

Article abstract of DOI:10.1016/S0008-6215(00)00200-7

Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1 → 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranoside (6), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1 → 3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside (14), 2-naphthylmethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1 → 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-galactopyranoside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1 → 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl-(1 → 6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-α-D-galactopyranoside (27). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00200-7

www.elsevier.nl/locate/carres
Carbohydrate Research 329 (2000) 287–300
Synthesis and glycan priming activity of acetylated
disaccharides
Arun K. Sarkar 1, Jillian R. Brown, Jeffrey D. Esko *
Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center,
Uni6ersity of California, San Diego, La Jolla, CA 92093, USA
Received 12 April 2000; accepted 22 June 2000
Abstract
Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using
orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1“4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b- -glucopyranoside (6), 2-naphthylmethyl 2-aceta-
mido-3,4,6-tri-O-acetyl-2-deoxy-b- -glucopyranosyl-(1“3)-2,4,6-tri-O-acetyl-b- -galactopyranoside (14), 2-naph-
thylmethyl2,3,4,6-tetra-O-acetyl-b- -galactopyranosyl-(1“3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-a- -galactopyran-
oside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b- -glucopyranosyl-(1“3)-2-acetamido-4,6-di-
O-acetyl-2-deoxy-a- -galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b- -glu-
copyranosyl-(1“6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-a- -galactopyranoside (27). These per-O-acetylated com-
D
-
D
D
D
D
D
D
D
D
D
pounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein
biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served
as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of
glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin. © 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Glycoside primers; Cell adhesion; Inhibitors; Sialyl Lewis X
occur amongst circulating tumor cells,
1. Introduction
platelets and endothelial cells, which may con-
tribute to metastasis [2,3]. Great interest exists
in finding inhibitors that block selectin–car-
bohydrate interactions since these compounds
might prove useful in treating pathological
conditions characterized by unwanted cellular
extravasation, as in chronic inflammatory dis-
eases, acute ischemic injury or tumor metasta-
sis [4].
Although the precise structure of the func-
tional carbohydrate ligand varies amongst the
different selectins and depends on the tissue
and organism under study [5], it typically con-
sists of one or more fucosylated and sialylated
glycans like those found on cell surface and
Sialyl Lewis X (sLeX), a common carbohy-
drate antigen expressed on leukocytes, medi-
ates cell adhesion to selectin receptors on
endothelial cells and platelets (reviewed in [1]).
The interaction initiates a cascade of events
that result in the eventual extravasation of
leukocytes from the circulation to lymph
nodes and inflamed tissue. Similar interactions
* Corresponding author. Tel.: +1-858-8221100; fax: +1-
858-5345611.
E-mail address: jesko@ucsd.edu (J.D. Esko).
1 Present address: North American Vaccine, Inc., 10150 Old
Columbia Road, Columbia, MD 21046, USA.
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00200-7

288
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
secreted mucins (Fig. 1). Attempts to interfere
with selectin-dependent adhesion have in-
volved the use of competitive ligands contain-
ing these sugars or analogs that bind the
carbohydrate recognition domain (see [6] and
references therein). Another approach is to
interfere with the biosynthesis of the oligosac-
charide chains. Towards this end, we have
demonstrated that per-O-acetylated disaccha-
rides such as 2-naphthylmethyl 2,3,4,6-tetra-
thetic disaccharides, their relative efficacy to-
wards priming oligosaccharide chains in U937
cells and their effect on selectin-dependent cell
adhesion.
2. Results and discussion
Synthesis of N-acetylglucosamine ana-
logues.—Reaction of 2-acetamido-3,4,6-tri-O-
O-acetyl-b-
amido-3,6-di-O-acetyl-2-deoxy-b-
ranoside (6) and 2-naphthylmethyl 2-acet-
amido - 3,4,6 - tri- O - acetyl-2-deoxy-b- -gluco-
pyranosyl-(1“3)-2,4,6-tri-O-acetyl-b- -gal-
D
-galactopyranosyl-(1“4)-2-acet-
acetyl-2-deoxy- -glucopyranosyl chloride with
D
D-glucopy-
2-naphthylmethanol [8] gave derivative 1, and
subsequent deprotection with 0.05 M sodium
methoxide afforded 2-naphthylmethyl 2-acet-
D
D
amido-2-deoxy-b- -glucopyranoside (2). In
D
actopyranoside (14) are taken up by a variety
of cells [6,7]. Endogenous carboxylesterases
remove the acetyl groups, generating com-
pounds that resemble intermediates in the
biosynthetic pathway (Galb1-4GlcNAc-R and
GlcNAcb1-3Gal-R). The glycosides give rise
to tri, tetra and pentasaccharides that are
secreted into the growth medium. The forma-
tion of these compounds diverts the assembly
of oligosaccharides away from endogenous
glycoproteins, thereby reducing the expression
of Lewis determinants on the cell surface.
Thus, these disaccharides inhibit adhesion me-
diated by selectin–sLeX interactions [6,7].
To extend these studies, we have now inves-
tigated three more per-O-acetylated 2-naph-
thylmethyl disaccharides that represent other
substructures found in vertebrate mucins.
Here, we report the synthesis of all five syn-
order to selectively glycosylate 2 at the 4-posi-
tion, the 3- and 6-position were selectively
benzoylated using benzoyl chloride at low
temperature [9], yielding 3 in 70% yield. Cou-
pling of 3 with ethyl-2,3,4,6-tetra-O-acetyl-1-
thio-b- -galactopyranoside as the glycosyl
D
donor [10–14] utilizing NIS-triflic acid medi-
ated activation gave low yields (5–10%). In
contrast, condensation in the presence of
bromine and silver trifluoromethanesulfonate
[15] afforded disaccharide, 4 in 32% yield.
After the removal of benzoate and acetate
groups by mild alkali treatment and subse-
quent per-O-acetylation, the target disaccha-
ride, 2-naphthylmethyl 2,3,4,6-tetra-O-acetyl-
b- -galactopyranosyl-(1“4)-2-acetamido-3,6-
D
di-O-acetyl-2-deoxy-b- -glucopyranoside (6)
D
was obtained in 84% yield (Scheme 1). Peaks
at l 4.50 (J 8.75 Hz) and 4.47 (J 7.25 Hz) in
1
the H NMR of 6 confirmed the b-glycosidic
linkage between the two sugars, and a peak at
l 5.44 (J 9.35 Hz) confirmed the presence of
the N-acetyl group of the GlcNAc residue in
the structure. 13C NMR signals at l 104.93
(C-1%), 99.41 (C-1) and 75.59 (C-4) supported
that the sugars were b-(1“4)-linked.
Fig. 1. Sialyl Lewis X (sLeX) occurs on the non-reducing end
of Ser/Thr-linked oligosaccharides.
Reaction of 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-a- -glucopyranosyl chloride with
D
known benzyl 2-O-benzoyl-4,6-O-benzyli-
dene-b-
D
-galactopyranoside [8,16,17] afforded
benzyl
2-acetamido-3,4,6-tri-O-acetyl-2-de-
oxy-b- -glucopyranosyl-(1“3)-2-O-benzoyl-
D
4,6-di-O-benzylidene-b- -galactopyranoside
D
(7). The removal of the benzylidene protecting
group under acidic conditions and purification
of the product by column chromatography
Scheme 1.

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
289
acetamidate [20] with 2-naphthylmethanol in
presence of trimethylsilyltrifluoromethanesul-
fonate (TMS triflate) in hexane–dichloro-
methane afforded 2-naphthylmethyl 3,4,6-tri-
O-acetyl-2-azido-2-deoxy-a- -galactopyrano-
D
side (15) in 61% yield. O-Deacetylation of 15
with 0.05 M sodium methoxide afforded 16.
Compound 16 was subsequently converted to
2-naphthylmethyl 2-azido-4,6-O-benzylidene-
2-deoxy-a- -galactopyranoside (17) by treat-
D
ment with benzaldehyde dimethylacetal in the
Scheme 2.
presence of p-toluenesulfonic acid. Reaction
of 17 with 2,3,4,6-tetra-O-acetyl-a- -galac-
D
topyranosyl trichloroacetamidate in the pres-
ence of TMS triflate [21] gave 2-naph-
thylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-
D
-galactopyranosyl-(1“3)-2-azido-
-galactopyran-
4,6-O-benzylidene-2-deoxy-a-
D
oside (18) in 71% yield. The benzylidene group
from 18 was removed with 80% acetic acid at
high temperature to afford 19. We adapted
the reaction scheme developed by Malik and
co-workers [22] to reduce the azide to an
amine (palladium-on-charcoal under refluxing
conditions in methanol). Subsequent acetyla-
tion with pyridine and acetic anhydride af-
forded the fully acetylated target disaccharide,
2-naphthylmethyl 2,3,4,6 - tetra - O - acetyl - b-
Scheme 3.
afforded 8. Subsequent peracetylation of 8
afforded benzyl 2-acetamido-3,4,6-tri-O-ace-
tyl-2-deoxy-b-
D
-glucopyranosyl-(1“3)-4,6-di-
-galactopyranoside
O-acetyl-2-O-benzoyl-b-
D
(9). Catalytic hydrogenation with palladium-
on-charcoal furnished 10, which was subse-
quently converted to the trichloroacetamidate
derivative 11 by treatment with 1,8-diazabicy-
clo[5.4.0]-undec-7-ene and trichloroacetoni-
trile. Reaction of 11 with 2-naphthylmethanol
[13,18] gave 2-naphthylmethyl 2-acetamido-
D
-galactopyranosyl-(1“3)-2-acetamido-4,6-
-galactopyranoside
di-O-acetyl-2-deoxy-a-
D
(20) in 70% yield (Scheme 3). Peaks at l 5.09
1
(J 4 Hz) and 4.57 (J 8 Hz) in the H NMR
3,4,6-tri-O-acetyl-2-deoxy-b-
syl-(1“3)-4,6-di-O-acetyl-2-O-benzoyl-b-
D
-glucopyrano-
spectrum of 20 confirmed the a-glycosidic
linkage between the GalNAc residue and the
aglycone and the b-glycosidic linkage between
the two sugars, respectively. A peak at l 5.64
D
-
galactopyranoside (12). Compound 12 was
treated with 0.1 M sodium methoxide to fur-
nish 13 and subsequently peracetylated with
pyridine and acetic anhydride to afford the
fully acetylated target disaccharide, 2-naph-
thylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-
1
(J 9 Hz) in the H NMR spectrum confirmed
the presence of the N-acetyl group of the
GalNAc residue, thereby confirming conver-
13
sion of N3 to NHAc. The C NMR spectrum
deoxy-b-
D
-glucopyranosyl-(1“3)-2,4,6-tri-O-
was in good accordance with the proposed
structure of compound 20.
acetyl-b-
D
-galactopyranoside (14) (Scheme 2).
1H and C NMR spectra were in good accor-
dance with the proposed structure of com-
pound 14.
13
Disaccharide derivative, 2-naphthylmethyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b- -
D
glucopyranosyl-(1“3)-2-azido-4,6-O-benzyli-
dene-2-deoxy-a- -galactopyranoside (21) was
obtained by treating 17 with 2-acetamido-
-glucopyranosyl
Synthesis of N-acetylgalactosamine analogs.
—For the synthesis of 2-naphthylmethyl gly-
cosides of N-acetylgalactosamine, we followed
the procedure described by Lemieux and Rat-
cliffe [19]. The reaction of 3,4,6-tri-O-acetyl-2-
D
3,4,6-tri-O-acetyl-2-deoxy-a-
D
chloride in the presence of mercuric cyanide
and mercuric chloride in 1:1 nitromethane–
benzene. The benzylidene group was removed
azido-2-deoxy-b- -galactopyranosyl trichloro-
D

290
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
galactopyranoside (24). Subsequently, 24 was
reacted with 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-a- -glucopyranosyl chloride in the
by treatment with 80% acetic acid at high
temperature to afford 2-naphthylmethyl 2-ac-
D
etamido-3,4,6-tri-O-acetyl-2-deoxy-b-
pyranosyl-(1“3)-2-azido-2-deoxy-a-
D
-gluco-
-galac-
presence of mercuric cyanide and mercuric
chloride to afford 25. The isopropylidene
group from 25 was removed with 70% acetic
acid at high temperature to afford 26. Com-
pound 26 was reacted with palladium-on-char-
coal and acetylated using pyridine and acetic
anhydride to furnish the fully acetylated target
disaccharide, 2-naphthylmethyl 2-acetamido-
D
topyranoside (22). Treatment of 22 with palla-
dium-on-charcoal and subsequent acetylation
with pyridine and acetic anhydride furnished
the desired target disaccharide, 2-naphthyl-
methy 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b -
D
- glucopyranosyl - (1“3) - 2 - acetamido-
-galactopyrano-
4,6-di-O-acetyl-2-deoxy-a-
D
3,4,6-tri-O-acetyl-2-deoxy-b-
D
-glucopyrano-
side (23) in 70% yield (Scheme 4). Peaks at l
5.09 (J 3.5 Hz) and 4.96 (J 8.5 Hz) in the H
1
syl-(1“6)-2-acetamido-3,4-di-O-acetyl-2-de-
oxy-a- -galactopyranoside (27) in 71% yield
D
NMR spectrum confirmed the a-glycosidic
linkage between the GalNAc residue and the
aglycone and the b-glycosidic linkage between
the two sugars, respectively. Two peaks at l
(Scheme 5). Peaks at l 5.00 (J 3.5 Hz) and
1
4.72 (J 8 Hz) in the H NMR spectrum con-
firmed the a-glycosidic linkage between the
GalNAc residue and the aglycone and the
b-glycosidic linkage between the two sugars,
respectively. Two peaks at l 5.61 (J 9.5 Hz)
1
6.04 (J 8.5 Hz) and 5.80 (J 8 Hz) in the H
NMR spectrum confirmed the presence of the
N-acetyl group of the GalNAc and GlcNAc
residues, respectively. 13C NMR signals at l
99.05 (C-1%), 96.97 (C-1) and 72.46 (C-3) sup-
port the proposed structure.
1
and 5.50 (J 8.5 Hz) in the H NMR spectrum
confirmed the presence of the N-acetyl group
of the GalNAc and GlcNAc residues, respec-
tively. The 13C NMR spectrum was in good
accordance with the proposed structure.
The disaccharide derivative, 2-naphthyl-
methyl
oxy-b-
2-acetamido-3,4,6-tri-O-acetyl-2-de-
-glucopyranosyl-(1“6)-2-azido-2-de-
Biological acti6ity of acetylated disaccha-
rides.—We have shown that cells take up and
rapidly deacetylate per-O-acetylated forms of
6 and 14 [6]. These disaccharides resemble
intermediates in the pathway and therefore act
as substrates, giving rise to fucosylated and
sialylated oligosaccharides. The priming of
oligosaccharides by per-O-acetylated forms of
20, 23 and 27 was therefore measured in com-
parison to 6 and 14. As shown in Table 1 and
previously [6], 6 stimulated the incorporation
of [6-3H]Fuc into oligosaccharides. Previous
studies showed that the glycan primed on 6
consisted entirely of LeX (Galb1-4(Fuca1-
3)GlcNAcb-R. In contrast, 14 primed
oligosaccharides containing Gal, Fuc and
sialic acid, including sLeX [6], and therefore
stimulated the incorporation of both [6-
3H]Fuc and [6-3H]Gal. The large increase in
incorporation of all three radioactive sugars
into oligosaccharides primed on 20 suggests
that a complex set of products were formed,
including GlcNAc-containing oligosaccha-
rides. Dennis and co-workers have shown that
several fucosylated and sialylated oligosaccha-
rides are assembled on benzyl a-GalNAcp
D
oxy-3,4-O-isopropylidene-a- -galactopyrano-
D
side (25) was obtained by first reacting 16 with
2,2-dimethoxypropane in presence of cam-
phorsulfonic acid to afford 2-naphthylmethyl
2-azido-2-deoxy-3,4-O-isopropylidene-a- -
D
Scheme 4.
Scheme 5.

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
291
Table 1
when the compound was fed to Colo 205 cells
[23]. Although detailed studies have not yet
been done on the oligosaccharides produced
on 20 in U937 cells, it seems likely that some
would be related to those primed on benzyl
a-GalNAcp since 20 resembles the next inter-
mediate in the pathway (Galb1-3GalNAca-R).
Neither 23 nor 27 stimulated the incorpora-
tion of precursors into oligosaccharides. The
failure of 23 to prime oligosaccharides sup-
ports the idea that extension of the b1-6
branch depends on prior formation of the core
structure (Galb1-3GalNAca-R) [24].
Oligosaccharide synthesis on acetylated primers in U937
cells a
Glycosides
[3H]Fuc
[3H]Gal
[3H]GlcN
cpm incorporated×10−3
Experiment I
None
6
4
9
14
16
84
77
72
b
14
20 b
53 b
Experiment II
None
20
10
35 b
12
19
52 b
21
68
160 b
78
23
To test whether the extent of priming by 20
was sufficient to inhibit the glycosylation of
endogenous glycoproteins, samples of radioac-
tively labeled cells were treated with
trichloroacetic acid to precipitate glycoconju-
gates and others macromolecules. As shown in
Table 2, 20 inhibited the incorporation of
labeled sugars into precipitated material with
potencies similar to that observed for 6 and
14. In contrast, 23 and 27 had no effect,
consistent with their negligible priming activ-
ity (Table 1). None of the compounds showed
any toxicity under these conditions, based on
the level of incorporation of [6-3H]thymidine
into DNA.
27
11
25
93
a U937 cells (2×105 cells) were incubated with the indicated
glycoside for 48 h in growth medium supplemented with 10
mCi/mL [6-3H]Fuc, 10 mCi/mL [6-3H]Gal, or 10 mCi/mL [6-
3H]GlcN. Radioactive oligosaccharides were isolated by Sep-
Pak C18 reversed-phase chromatography and quantitated (see
Section 3). The data represents the average of duplicate
determinations that varied by B20%.
b Indicates values that were significantly different from the
controls in which no compound was added.
Table 2
Glycoside inhibition of glycosylation of endogenous glyco-
proteins in U937 cells a
To test if inhibiting glycosylation had any
effect on cell adhesion, treated U937 cells were
challenged to adhere to E-selectin expressed
on TNF-a-activated human umbilical vein en-
dothelial cells. As shown in Fig. 2, adhesion
was dependent on prior stimulation of the
endothelial cells with cytokine and on sialic
acids expressed on the surface of U937 cells.
Treatment of U937 cells with 50 mM of 6, 14
or 20 showed reduction in adhesion and the
effect was dose dependent [6]. As expected
from the results shown in Tables 1 and 2, 23
and 27 had no significant effect on adhesion.
These data support the idea that per-O-
acetylated disaccharides provide a rich source
of potential inhibitors by acting as biosyn-
thetic decoys. One advantage is that these
compounds have inhibitory effects in the
micromolar range, whereas other compounds
such as aryl N-acetyl-galactosaminides require
millimolar amounts to observe comparable ef-
fects [23,25,26]. This difference in effectiveness
is not related to the aglycone, as the per-O-
Glycoside
[3H]Thy
[3H]Fuc
[3H]Gal
[3H]GlcN
cpm×10−3
Experiment I
None
6
235
222
240
38
370
nd
nd
nd
22 b
24 b
240 b
235 b
14
Experiment II
None
20
23
27
192
192
189
178
47
291
194
26 b
43
164 b
272
147 b
194
44
277
186
a U937 cells (2×105 cells) were incubated with or without
glycosides (50 mM) for 28 h, and then the cells were labeled
with 10 mCi/mL [6-3H]Fuc, 10 mCi/mL [3H]Gal, 20 mCi/mL
[6-3H]GlcN, or 1 mCi/mL [3H-methyl]thymidine for another
20 h. Cellular glycoconjugates were precipitated with 10%
trichloroAcOH, washed once with 2% trichloroAcOH, and
dissolved in a small volume of 0.1 M sodium hydroxide. A
portion of each sample was counted by liquid scintillation
spectrometry. Each experiment was performed in duplicate,
and the average value varied by 510%; nd, not determined.
b Indicates values that were significantly different from
controls.

292
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
acetylated or free GalNAca-napthalen-
emethanol derivatives have the same dose
profile as GalNAca-benzyl (data not shown).
Presumably the enhanced efficacy of the disac-
charides reflects their greater similarity to nat-
ural intermediates in the pathway and higher
affinities for the relevant transferases. The fact
that some compounds do not act as primers
(e.g., 23) presumably reflects the restricted or-
der of reactions during the assembly process.
Thus, disaccharides like the ones reported
here allow one to probe the biosynthetic path-
way in vivo and to make correlations with
substrate specificity studies of the individual
enzymes in vitro. In other cases (e.g., 27), the
failure to prime oligosaccharides may merely
reflect the restricted repertoire of glycosyl-
transferases expressed by U937. Further stud-
ies of the compounds in other cell lines should
provide insight into this problem.
tubes, Wilmad 528-PP). A Varian 5-mm
pulsed-field-gradient inverse-detection (PFG-
ID) probe was used for all experiments. A
SUN Microsystems Ultra-10 computer run-
ning Varian’s VNMR software (version 6.1B)
controlled data acquisition. Chloroform was
used as internal standard for chemical shift
1
calibration; the H chemical shift of CHCl3
was set to 7.262 ppm, the 13C chemical shift of
CDCl3 to 77.0 ppm. Two-dimensional (2D) 1H
double-quantum-filtered COSY data sets were
collected in phase-sensitive mode. In each of
the experiments, 256 FIDs of 2048 complex
data points were acquired. The data were
processed with a squared sine bell function
applied in the t2 dimension and a shifted
squared sine bell function and zero-filling ap-
plied in the t1 dimension. Two-dimensional
heteronuclear
single-quantum
coherence
(HSQC) data sets, providing one-bond 1H, 13C
correlation maps, were collected in phase-sen-
sitive mode, with BIRD nulling of H–12C
1
signal components during the preparation pe-
3. Experimental
13
riod and C GARP decoupling during acqui-
sition. In each of the experiments, 512 FIDs of
2048 complex data points were acquired, with
32 or 64 scans per t1 increment. HSQC data
were processed with a squared sine bell func-
tion applied in the t2 dimension and a line
broadening function and zero-filling applied in
General methods.—Optical rotations were
measured at ambient temperature with an Au-
topol III automatic polarimeter. NMR experi-
ments were carried out on a Varian Unity
Inova 500 spectrometer, at 25 °C in CDCl3
(sample volumes of 0.6–0.7 mL in 5-mm
1
the t1 dimension. H NMR spectra (500 MHz)
of intermediates were recorded at 25 °C on a
Bruker AM 500 spectrometer. Selected NMR
data for intermediates are reported in Table 3,
and the spectra were interpreted by compari-
son of the chemical shifts to those obtained
with the parent compound. Elemental analy-
ses were performed by Robertson Microlit
Laboratories, Inc. (Madison, NJ). All reaction
products were analyzed by analytical thin-
layer chromatography (TLC) on Silica Gel
60-F254 (E. Merck) plates and detection by UV
light or by charring with 5% (v/v) H2SO4 in
EtOH. Column chromatography was per-
formed on Silica Gel 60-F254 (Aldrich). Molec-
Fig. 2. Adhesion of U937 cells to activated HUVEC. U937
cells (5×105 cells) were radiolabeled for 48 h at 37 °C with
[6-3H]thymidine and the per-O-acetylated disaccharides at the
indicated concentrations. The cells were then challenged to
adhere to TNF-a-activated HUVEC, and the total number of
adherent cells were measured (see Section 3). Control incuba-
tions with untreated U937 cells were carried out in the
absence of TNF-a (−) or with TNF-a (+) to demonstrate
the dependence on E-selectin activation. Another sample of
U937 cells was treated with sialidase to destroy Lewis anti-
gens on the cell surface. Each measurement is the average of
duplicate determinations that varied by 515%.
,
ular sieves (4 A) were heated to 180 °C for 24
h prior to use. All solvents were distilled prior
to use and stored over molecular sieves. All
evaporations were carried out at 40–50 °C
under reduced pressure, unless otherwise
noted. Solvents used for TLC A, 2:1 EtOAc–

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
293
3H]fucose (83 Ci/mmol), [6-3H]galactose (32
Ci/mmol), [6-3H]glucosamine (33.3 Ci/mmol),
or 1 mCi/mL [3H-methyl]thymidine (83 Ci/
mmol) (NEN Life Science Products, Inc.,
Boston, MA.).
Table 3
1
Selected H NMR data of synthetic intermediates
Compounds Chemical shifts (l)
1
2
3
4
7.80–7.40 (m, 7 H, ArH), 5.32 (d, 1 H,
J2,NH 8.9 Hz, NꢀH), 4.67 (d, 1 H, J1,2 8.5
Hz, H-1), 2.10–1.90 (12 H, CH3CO)
7.80–7.40 (m, 7 H, ArH), 5.0 (1 H, NꢀH),
4.41 (d, 1 H, J1,2 8.5 Hz, H-1), 1.84 (s, 3 H,
CH3CO)
8.10–7.39 (m, 17 H, ArH), 5.5 (d, 1 H,
J2,NH 9.2 Hz, NꢀH), 4.76 (d, 1 H, J1,2 8.2
Hz, H-1), 1.79 (s, 3 H, CH3CO)
8.08–7.36 (m, 17 H, ArH), 5.62 (d, 1 H,
J2,NH 9.5 Hz, NꢀH), 4.57 (d, 1 H, J1%,2% 7.9
Hz, H-1%), 4.62 (d, 1 H, J1,2 7.2 Hz, H-1),
2.00–1.90 (5 s, 15 H, CH3CO)
8.00–7.32 (m, 10 H, ArH), 5.32 (d, 1 H,
J2,NH 9.2 Hz, NꢀH), 5.04–4.89 (m, 2 H,
H-1% and H-4%), 4.40 (d, 1 H, J1,2 8.2 Hz,
H-1), 2.12–1.89 (12 H, CH3CO)
2-Naphthylmethyl 2,3,4,6-tetra-O-acetyl-i-
D
-galactopyranosyl-(1“4)-2-acetamido-3,6-
di-O-acetyl-2-deoxy-i- -glucopyranoside (6).
D
,
—To a suspension of molecular sieves (4 A, 5
g) in CH2Cl2 (100 mL) was added silver trifl-
uoromethanesulfonate (16 g, 62 mmol). The
mixture was stirred for 1 h at rt in the dark
under argon. The reaction mixture was cooled
to 0 °C, and 2-naphthylmethanol (12 g, 76
mmol) was added with stirring, followed by
the addition of a soln of 2-acetamido-3,4,6-tri-
8
O-acetyl-2-deoxy-a- -glucopyranosyl chloride
D
(10 g, 27 mmol) in anhyd CH2Cl2 (50 mL)
dropwise during 1 h. Stirring was continued
for 2 h at 0 °C and the reaction was stopped
12
8.01–7.26 (m, 12 H, ArH), 5.51 (d, 1 H,
J2,NH 9.5 Hz, NꢀH), 4.99 (d, 1 H, J1%,2%
8
Hz, H-1%), 4.42 (d, 1 H, J1,2 8.2 Hz, H-1),
2.10–1.92 (18 H, CH3CO)
and filtered through
a
bed of Celite.
Dichloromethane was added (150 mL), and
the organic layer washed successively with a
soln of 5% sodium thiosulfate, water and cold
satd NaHCO3 soln. The organic layer was
dried over Na2SO4, filtered and evaporated to
dryness. Precipitation of the reaction with
ether yielded the monosaccharide, 2-naphthyl-
methyl 2-acetamido - 3,4,6 - tri - O - acetyl - 2-
16
17
18
7.8–7.4 (m, 7 H, ArH), 5.01 (1 H, J1,2 3.1
Hz, H-1), 3.75 (dd, 1 H, H-2)
7.86–7.37 (m, 12 H, ArH), 5.56 (s, 1 H,
benzylidene), 5.16 (d, 1 H, J1,2 3.5 Hz, H-1)
7.86–7.32 (m, 12 H, ArH), 5.54 (s, 1 H,
benzylidene), 5.18 (d, 1 H, J1,2 3.5 Hz, H-1),
4.78 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 2.14–1.98
(4 s, 12 H, CH3CO)
7.84–7.32 (m, 7 H, ArH), 5.16 (d, 1 H, J1,2
3.5 Hz, H-1), 4.78 (d, 1 H, J1%,2% 7.9 Hz,
H-1%), 2.14–1.96 (4 s, 12 H, CH3CO)
7.86–7.46 (m, 12 H, ArH), 5.52 (s, 1 H,
benzylidene), 5.46 (d, 1 H, J2,NH 9.1 Hz,
NꢀH), 5.04 (d, 1 H, J1,2 3.1 Hz, H-1), 4.78
(d, 1 H, J1%,2% 8.0 Hz, H-1%), 2.1–1.9 (4 s, 12
H, CH3CO)
19
21
deoxy-b- -glucopyranoside (1) (9.5 g, 71%). Rf
D
1
0.14 (solvent A); [h]D −5° (c 0.2, CHCl3); H
NMR (CDCl3): Table 3; Anal. Calcd for
C25H29NO9: C, 61.59; H, 6.00; N, 2.87. Found:
C, 61.52; H, 6.15; N, 2.64.
Compound 1 (5 g, 10 mmol) was stirred at
rt with 0.05 M CH3ONa in MeOH (50 mL) for
3 h. The reaction mixture was neutralized with
AcOH and concd to dryness. The solid residue
was resuspended in MeOH (25 mL) and kept
in the cold for 2 h, whereby the precipitate,
22
7.82–7.42 (m, 7 H, ArH), 5.65 (d, 1 H,
NꢀH), 5.03 (d, 1 H, J1,2 3.2 Hz, H-1), 4.87
(d, 1 H, J1%,2% 8.1 Hz, H-1%), 2.05–1.98 (4 s,
12 H, CH3CO)
7.84–7.48 (m, 7 H, ArH), 5.05 (d, 1 H, J1,2
3.25 Hz, H-1), 1.36, 1.22 (2s, 6 H, 2-Pr)
7.86–7.48 (m, 7 H, ArH), 5.46 (d, 1 H,
J2,NH 8.4 Hz, NꢀH), 5.06 (d, 1 H, J1,2 3.5
Hz, H-1), 4.62 (d, 1 H, J1%,2% 8.0 Hz, H-1%),
2.12–1.98 (4 s, 12 H, CH3CO), 1.36 and
1.22 (2 s, 6 H, 2-Pr)
24
25
2-naphthylmethyl 2-acetamido-2-deoxy-b- -
D
glucopyranoside (2) (3 g, 81%) was collected
1
by filtration. Rf 0.4 (solvent C); H NMR
(CDCl3): Table 3; Anal. Calcd for C19H23NO6:
C, 63.15; H, 6.41; N, 3.88. Found: C, 63.12; H,
6.24; N, 3.65.
Compound 2 (2 g, 6 mmol) was dissolved in
pyridine (40 mL), and then cooled to −40 °C.
Benzoyl chloride (1.4 mL) was diluted with
pyridine (4 mL), and the mixture was added to
26
7.86–7.49 (m, 7 H, ArH), 5.44 (d, 1 H,
J2,NH 8.8 Hz, NꢀH), 5.04 (d, 1 H, J1,2 3.2
Hz, H-1), 4.64 (d, 1 H, J1%,2% 8.2 Hz, H-1%),
2.11–1.88 (4 s, 12 H, CH3CO)
hexane; B, 3:1 EtOAc–hexane; C, 9:1 CHCl3–
MeOH; D, 13:6:1 CHCl3–MeOH–water. [6-

294
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
tected disaccharide, 2-naphthylmethyl b-
galactopyranosyl-(1“4)-2-acetamido-2-de-
-glucopyranoside (5) (0.22 g, 84%); Rf
0.37 (solvent D); [h]D −5° (c 0.2, MeOH).
Anal. Calcd for C25H33NO11: C, 57.35; H,
6.35; N, 2.68. Found: C, 57.07; H, 6.55; N,
2.44.
Compound 5 (0.1 g, 0.2 mmol) was per-O-
acetylated by stirring with 1:1 pyridine–Ac2O
(1 mL) for 16 h at rt. The reaction mixture
was concd to dryness, and the residue was
purified by column chromatography (9:1
EtOAc–hexane) to yield the fully acetylated
target disaccharide, 2-naphthylmethyl 2,3,4,6-
D
-
the soln of compound 2 dropwise during 2 h
with stirring, maintaining the temperature at
−40 °C. The reaction mixture was stirred for
a further 1 h at −40 °C and then allowed to
warm up to rt while stirring for 2 h. The
excess benzoyl chloride was decomposed by
the addition of MeOH (0.4 mL). The reaction
mixture was concd to dryness and purified by
column chromatography (CHCl3 and 5% ace-
tone in CHCl3) yielding the monosaccharide,
2-naphthylmethyl 2-acetamido-3,6-di-O-ben-
oxy-b-
D
zoyl-2-deoxy-b- -glucopyranoside (3) (2.2 g,
D
70%); Rf 10.35 (solvent A); [h]D −1° (c 0.2,
CHCl3); H NMR (CDCl3): Table 3; Anal.
Calcd for C33H31NO8: C, 69.58; H, 5.49; N,
2.46. Found: C, 69.57; H, 5.42; N, 2.33.
tetra-O-acetyl-b-
D
-galactopyranosyl-(1“4)-
-glu-
2-acetamido-3,6-di-O-acetyl-2-deoxy-b-
D
Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-b-
D
-gal-
copyranoside (6) (0.127 g, 84%). Rf 0.24 (sol-
vent B); [h]D −2° (c 0.2, CHCl3); H NMR
1
actopyranoside (1.3 g, 3.2 mmol) and molecu-
,
lar sieves (4 A, 2.5 g) were added to a soln of
(CDCl3): l 7.84–7.39 (m, 7 H, ArH), 5.44 (d,
1 H, J2,NH 9.35, N-H), 5.35 (d, 1 H, H-3%),
5.12–5.08 (dd, 1 H, H-2%), 5.03–4.95 (m, 3 H,
H-3, H-3% and CH2), 4.74 (dd, 1 H, CH2), 4.56
(d, 1 H, H-6a/b), 4.50 (d, 1 H, J1%,2% 8.75 Hz,
H-1%), 4.47 (d, 1 H, J1,2 7.25 Hz, H-1), 4.19–
4.13 (m, 2 H, H-2 and H-6a/b), 4.12–4.09 (m,
2 H, H-6%a and H-6%b), 3.88 (dd, 1 H, H-5%),
3.83 (dd, 1 H, H-4), 3.62–3.59 (m, 1 H, H-5),
2.14, 2.13, 2.05, 2.04, 2.03, 1.96, 1.94 (7s, 21
H, CH3CO); 13C NMR (CDCl3): l 169.32–
170.66 (CꢁO), 134.25, 133.13, 133.03, 128.29,
127.83, 127.12, 126.87, 126.35, 126.19, 125.65
(10 C, ArC), 100.93 (C-1%), 99.41 (C-1), 75.59
(C-4), 72.70 (C-5), 72.33, 70.82 (2 C), 70.50,
69.00 (C-2%), 66.55 (C-4%), 62.36 (C-6), 60.75
(C-6%), 53.04 (C-2), 29.68 (CH3CON), 20.92–
20.28 (6 C, CH3COO). Anal. Calcd for
C37H45NO17: C, 57.29; H, 5.85; N, 1.81.
Found: C, 57.11; H, 5.68; N, 1.62.
compound 3 (1 g, 2 mmol) in CH2Cl2 (25 mL).
The reaction mixture was allowed to stir at rt
for 2 h under argon. Silver trifluoromethane-
sulfonate (1.8 g, 6.8 mmol) was added, and
stirring was continued for 15 min, then Br2
(0.1 mL) was added, and the reaction mixture
was stirred for a further 2 h under argon.
Triethylamine (0.5 mL) was added to quench
the reaction, and the reaction mixture was
filtered through a bed of Celite. Dichloro-
methane was added (100 mL), and the organic
layer was washed successively with cold 5%
sodium thiosulfate, cold satd NaHCO3 and
cold water. The reaction was dried over
Na2SO4, filtered and concd to dryness. The
residue was purified by column chromatogra-
phy (increasing gradient of EtOAc in hexane
30“50“60%) yielding the disaccharide inter-
mediate, 2-naphthylmethyl 2,3,4,6-tetra-O-
acetyl-b-
D
-galactopyranosyl-(1“4)-2-aceta-
-glucopy-
2-Naphthylmethyl 2-acetamido-3,4,6-tri-O-
mido-3,6-di-O-benzoyl-2-deoxy-b-
D
acetyl-2-deoxy-i-
2,4,6-tri-O-acetyl-i-
—Benzyl 2-O-benzoyl-4,6-O-benzylidene-b- -
D
-glucopyranosyl-(1“3)-
ranoside (4) (0.5 g, 32%); Rf 0.29 (solvent A);
[h]D −1° (c 0.3, CHCl3); H NMR (CDCl3):
D
-galactopyranoside (14).
1
D
Table 3; Anal. Calcd for C47H49NO17: C,
62.73; H, 5.49; N, 1.56. Found: C, 62.44; H,
5.67; N, 1.46.
Compound 4 (0.45 g, 0.50 mmol) was
stirred with 0.1 M CH3ONa in MeOH (15
mL) for 8 h, after which time the reaction was
neutralized with IR-120 (H+) cation-exchange
resin. The resin was filtered, and the mixture
was concd to dryness to give the fully depro-
galactopyranoside (1.3 g, 2.7 mmol) was dis-
solved in 1:1 nitromethane–benzene (45 mL),
and approximately one-third of the solvent
was distilled off. The reaction mixture was
,
cooled to 35–40 °C, molecular sieves (4 A, 2
g) were added, and the reaction mixture was
stirred. Mercuric cyanide (1.4 g, 5.5 mmol)
and mercuric bromide (1.8 g, 5.0 mmol) were
added, and the reaction mixture was stirred

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
295
undec-7-ene (0.15 mL) and CCl3CN (0.4 mL)
were added. The reaction mixture was stirred
at rt for 1 h, concd to dryness and subjected
to column chromatography (2:1 EtOAc–hex-
ane containing 0.1% Et3N) to yield 2-acetam-
for 1 h at 35–40 °C under argon and 2-acet-
amido-3,4,6-tri-O -acetyl-2-deoxy-a- -gluco-
pyranoside (1.5 g, 4.1 mmol) was added. The
reaction mixture was stirred for 16 h at 35–
40 °C under argon, and then additional mer-
curic cyanide (0.6 g, 2.4 mmol), mercuric
bromide (0.8 g, 2.2 mmol) and 2-acetamido-
D
ido - 3,4,6 - tri - O - acetyl - 2 - deoxy-b-
pyranosyl-(1“3)-4,6-di-O-acetyl-2-O-benz-
-galactopyranosyl trichloroacetamidate
D-gluco-
3,4,6-tri-O-acetyl-2-deoxy-a-D-glucopyranosyl
oyl-a-D
chloride (0.8 g, 2.1 mmol) were added and
stirred for another 20 h at 35–40 °C. The
reaction mixture was filtered and diluted with
benzene (100 mL), and the organic layer was
washed with satd NaHCO3, satd KI, and wa-
ter. The organic layer was dried over Na2SO4,
filtered and concd to dryness. The crude reac-
tion mixture of disaccharide 7 was used di-
rectly in the next step, whereby the reaction
mixture was treated with 80% AcOH at 80 °C
for 2 h. The solvent was concd to dryness and
precipitation with ether yielded benzyl 2-acet-
(11) (0.5 g, 82%). To a soln of 11 (0.5 g, 0.6
mmol) and 2-naphthylmethanol (0.4 g, 2.5
mmol) in anhyd CH2Cl2 (10 mL) was added
toluene (10 mL). The reaction mixture was
,
stirred with molecular sieves (4 A, 2.5 g) for 1
h under argon at rt. The reaction mixture was
cooled to −10 °C, and a soln of trimethylsilyl
trifluoromethanesulfonate (TMS triflate) (0.5
M soln in toluene, 0.5 mL) was added with
stirring at −10 °C for 1 h. The reaction mix-
ture was then neutralized with Et3N, filtered
and concd to dryness. The residue was
purified by column chromatography (increas-
ing gradient of EtOAc in hexane 33“50“
66“75%) to yield 2-naphthylmethyl 2-aceta-
amido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-gluco-
pyranosyl-(1“3)-2-O-benzoyl-b- -galacto-
D
pyranoside (8) (0.77 g, 41%); Rf 0.25 (Solvent
B); [h]D −5° (c 0.2, CHCl3); 1H NMR
(CDCl3): Table 3; Anal. Calcd for C34H41-
NO15: C, 58.03; H, 5.87; N, 1.99. Found: C,
57.71; H, 5.72; N, 2.05.
mido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-glucopy-
ranosyl-(1“3)-4,6-di-O-acetyl-2-O-benzoyl-
b- -galactopyranoside (12) (0.4 g, 81% from
D
11): Rf 0.25 (solvent B); [h]D −2° (c 0.2,
1
Compound 8 (0.8 g, 1.1 mmol) was acety-
lated by stirring in 1:1 pyridine–Ac2O (10 mL)
at rt overnight. The reaction mixture was
evaporated to dryness, and the resulting
residue was subjected to column chromatogra-
phy (19:1 CHCl3–MeOH) to yield the disac-
charide, benzyl 2-acetamido-3,4,6-tri-O-acetyl-
CHCl3); H NMR (CDCl3): Table 3; Anal.
Calcd for C42H47NO17: C, 60.21; H, 5.65; N,
1.67. Found: C, 60.28; H, 6.08; N, 1.72.
Compound 12 (0.3 g, 0.4 mmol) was O-
deacetylated using 0.1 M CH3ONa in MeOH
(5 mL), and after standard work up yielded
2-naphthylmethyl 2-acetamido-2-deoxy-b-
D
-
2-deoxy-b-
D
-glucopyranosyl-(1“3)-4,6-di-O-
-galactopyranoside (9)
glucopyranosyl-(1“3)-b- -galactopyranoside
D
acetyl-2-O-benzoyl-b-
D
(13) (0.18 g, 94%); Rf 0.4 (solvent D), [h]D
−2° (c 0.4, 0.05% Me2SO in MeOH). Anal.
Calcd for C25H33NO11: C, 57.35; H, 6.35; N,
2.68. Found: C, 57.08; H, 6.59; N, 2.41.
(0.75 g, 89%); Rf 0.24 (solvent A), [h]D 0° (c
0.2, CHCl3); Anal. Calcd for C38H45NO17: C,
57.93; H, 5.76; N, 1.78. Found: C, 58.23; H,
6.04; N, 1.78.
Compound 13 (0.15 g, 0.29 mmol) was
acetylated with 1:1 pyridine–Ac2O (5 mL),
after which the solvent was concd to dryness.
Column chromatography (4:1 EtOAc–hex-
ane) yielded the fully acetylated target disac-
charide, 2-naphthylmethyl 2-acetamido-3,4,6-
A soln of 9 (0.75 g, 0.95 mmol) in AcOH
(10 mL) was hydrogenated in presence of 10
wt.% Pd–C (0.3 g) by shaking under H2 (52
psi) overnight at rt. The reaction mixture was
filtered and concd to dryness to give 2-acetam-
ido - 3,4,6 - tri - O - acetyl - 2 - deoxy - b-
pyranosyl-(1“3)-4,6-di-O-acetyl-2-O-benz-
oyl- -galactopyranoside (10). Crude com-
D
-gluco-
tri - O - acetyl - 2 - deoxy - b -
D
- glucopyranosyl-
-galactopyrano-
(1“3)-2,4,6-tri-O-acetyl-b-
D
D
side (14) (0.2 g, 90%); Rf 0.18 (solvent C), [h]D
1
pound 10 (0.5 g, 0.7 mmol) was used directly
in the next step. It was dissolved in anhyd
CH2Cl2 (10 mL), and 1,8-diazabicyclo[5.4.0]-
−2° (c 0.2, CHCl3); H NMR (CDCl3): l
7.85–7.39 (m, 7 H, ArH), 5.50 (dd, 1 H,
H-3%), 5.41 (d, 1 H, J2,NH 9.6 Hz, N-H), 5.36

296
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
with stirring. The reaction was allowed to stir
for 5 h at rt. Triethylamine (0.5 mL) was
added to quench the reaction, the solvent was
concd to dryness, and the residue was sub-
jected to column chromatography (1:1
EtOAc–hexane) to yield 2-naphthylmethyl 2-
(dd, 1 H, H-4), 5.24 (dd, 1 H, H-2), 5.04–4.99
(m, 3 H, H-1%, H-4%, CH2), 4.79 (d, 1 H, CH2),
4.44 (d, 1 H, J1,2 7.7 Hz, H-1), 4.30 (dd, 1 H,
H-6%a/b), 4.17–4.12 (m, 2 H, H-6a and H-6b),
4.07–4.04 (m, 1 H, H-6%a/b), 3.79–3.77 (m, 2
H, H-3 and H-5), 3.64–3.60 (dd, 1 H, H-5%),
3.31–3.25 (dd, 1 H, H-2%), 2.13, 2.10, 2.08,
2.05, 2.04, 2.00, 1.89 (7s, 21 H, CH3CO). 13C
NMR (CDCl3): l 170.72–169.48 (CꢁO),
134.18, 133.12, 133.03, 128.28, 127.82, 127.73,
126.87, 126.33, 126.17, 125.69 (10 C, ArC),
99.59 (C-1%), 99.22 (C-1), 75.98, 71.58 (C-5%),
71.25 (C-3%), 71.10, 70.85 (C-2), 70.46, 68.74,
69.22 (C-4), 62.09 (C-6), 61.24 (C-6%), 56.17
(C-2%), 23.30 (CH3CON), 21.07–20.64 (6 C,
CH3COO). Anal. Calcd for C37H45NO17: C,
57.29; H, 5.85; N, 1.81. Found: C, 57.28; H,
6.08; N, 1.72.
azido-4,6-O-benzylidene-2-deoxy-a- -galacto-
D
pyranoside (17) (1.05 g, 83%); Rf 0.68 (solvent
1
B); [h]D +16° (c 0.2, CHCl3); H NMR
(CDCl3): Table 3; Anal. Calcd for C24H25-
N3O5: C, 66.19; H, 5.79; N, 9.65. Found: C,
66.51; H, 5.53; N, 9.68.Compound 17 (0.4 g,
0.9 mmol) was dissolved in anhyd CH2Cl2 (15
,
mL) and stirred with molecular sieves (4 A,
2.5 g) under argon for 30 min. 2,3,4,6-tetra-O-
acetyl-a-
D
-galactopyrosyl
trichloroacetami-
date (0.6 g, 1.1 mmol) was added, followed by
toluene (10 mL). After stirring for 10 min at
rt, the reaction mixture was cooled to
−10 °C, and a 0.5 M soln of TMS triflate
(final concentration of TMS triflate in the
reaction mixture was 0.01 M) was added. The
reaction mixture was stirred for a further 1 h
at −10 °C under argon. Triethylamine (0.5
mL) was added to quench the reaction, and it
was filtered through Celite and concd to dry-
ness. The residue was purified by column
chromatography using an increasing gradient
of EtOAc in hexane (25“33“50%) to yield
2-Naphthylmethyl 2,3,4,6-tetra-O-acetyl-i-
D
-galactopyranosyl-(1“3)-2-acetamido-4,6-
di-O-acetyl-2-deoxy-h-
(20).—To a soln of 3,4,6-tri-O-acetyl-2-azido-
2-deoxy-b- -galactopyranosyl trichloroac-
D
-galactopyranoside
D
etamidate (4.0 g, 8.4 mmol) in CH2Cl2 (70
mL) was added 2-naphthylmethanol (2.7 g,
17.5 mmol). The reaction mixture was cooled
to −20 °C, and a soln of TMS triflate in
CH2Cl2 (to a final concentration of 0.05 M in
the reaction mixture) was added. The reaction
mixture was stirred for 30 min at −20 °C,
neutralized with Et3N, and concd to dryness.
Column chromatography (9:1 toluene–ace-
tone) gave 2-naphthylmethyl 3,4,6-tri-O-ace-
2-naphthylmethyl 2,3,4,6-tetra-O-acetyl-b-
D
-
galactopyranosyl-(1“3)-2-azido-4,6-O-benzy-
lidene-2-deoxy-a-
D
-galactopyranoside
(18)
(0.5 g, 71%); Rf 0.61 (solvent B), [h]D +5° (c
1
tyl-2-azido-2-deoxy-a-D-galactopyranoside
0.2, CHCl3); H NMR (CDCl3): Table 3;
(15) (2.4 g, 61%); Rf 0.72 (solvent C). Com-
pound 15 (2.2 g, 4.7 mmol) was used directly
in the next step, whereby the reaction mixture
was O-deacetylated with 0.02 M CH3ONa in
MeOH (25 mL). After standard work-up pro-
cedure, the reaction mixture was concd to
dryness, and precipitation from ice-cold
MeOH gave 2-naphthylmethyl 2-azido-2-
Anal. Calcd for C38H43N3O4: C, 59.60; H,
5.66; N, 5.49. Found: C, 58.96; H, 5.56; N,
5.33.
Compound 18 (0.4 g, 0.5 mmol) was treated
with 80% AcOH for 2 h at 80 °C. The solvent
was evaporated off, and the resulting immo-
bile oil residue was subjected to column chro-
matography (CHCl3) to give 2-naphthyl-
deoxy-a-
D
-galactopyranoside (16) (1 g, 62%).
methyl 2,3,4,6-tetra-O-acetyl-b-
D
-galacto-pyran-
Rf 0.4 (solvent C), [h]D +24° (c 0.2, CHCl3);
1H NMR (CDCl3): Table 3; Anal. Calcd for
C17H19N3O5: C, 59.12; H, 5.55; N, 12.17.
Found: C, 58.41, H, 5.61; N, 11.77.
osyl-(1“3)-2-azido-2-deoxy-a-
D
-galactopy-
ranoside (19) (0.29 g, 83%); Rf 0.37 (solvent
B), [h]D +5° (c 0.2, CHCl3); 1H NMR
(CDCl3): Table 3; Anal. Calcd for C31H37-
N3O4: C, 55.11; H, 5.52; N, 6.22. Found: C,
55.21; H, 5.92; N, 5.98.
To a soln of compound 16 (1 g, 3 mmol) in
N,N-dimethylformamide (DMF, 15 mL) was
added benzaldehyde dimethylacetal (1 mL)
and p-toluenesulfonic acid (0.2 g, 1.1 mmol)
To a soln of compound 19 (0.2 g, 0.3 mmol)
in MeOH (5 mL) was added 20 wt.% palla-

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
297
overnight at 40 °C under argon. The reaction
mixture was filtered through Celite and
washed successively with cold satd NaHCO3,
satd KI, and water. The organic layer was
concd to dryness, and column chromatogra-
phy (1:1 EtOAc–hexane) gave 2-naphthyl-
dium hydroxide–charcoal (0.04 g) and hydra-
zine (0.15 mL). The reaction mixture was
heated under gentle reflux for 2 h, after which
time the solvent was concd to dryness and the
residue was per-O-acetylated with 1:1 pyri-
dine–Ac2O (2 mL). The reaction mixture was
concd to dryness and purified by column chro-
matography with increasing gradient of
EtOAc in hexane (33“50“66“75%) to give
the fully acetylated target disaccharide, 2-
methyl
2-acetamido-3,4,6-tri-O-acetyl-2-de-
-glucopyranosyl-(1“3)-2-azido-4,6-
oxy-b-
D
O-benzylidene-2-deoxy-a- -galactopyranoside
D
(21) (0.17 g, 48%); Rf 0.52 (solvent B); [h]D
1
naphthylmethyl
2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl-(1“3)-2-acetamido-4,6-di-
D-
+2° (c 0.2, CHCl3); H NMR (CDCl3): Table
3; Anal. Calcd for C38H44N4O13: C, 59.68; H,
5.80; N, 7.33. Found: C, 59.92; H, 5.56; N,
7.55.
Compound 21 (0.1 g, 0.2 mmol) was treated
with 80% AcOH at 80 °C for 2 h. The solvent
was concd to dryness, and the residue was
subjected to column chromatography (19:1
CHCl3–MeOH) to give 2-naphthylmethyl 2-
O-acetyl-2-deoxy-a-
D
-galactopyranoside (20)
(0.16 g, 70%); Rf 0.30 (solvent B), [h]D 0° (c
1
0.2, CHCl3); H NMR (CDCl3): l 7.89–7.43
(m, 7 H, ArH), 5.64 (d, 1 H, J2,NH 9 Hz,
N-H), 5.40 (dd, 1 H, H-4), 5.33 (dd, 1 H,
H-4%), 5.12 (dd, 1 H, H-2%), 5.09 (d, 1 H, J1,2
Hz, H-1), 4.92 (dd, 1 H, H-3%), 4.85 (d, 1 H,
CH2), 4.61 (d, 1 H, CH2), 4.57 (d, 1 H, J1%,2%
4
8
acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-glu-
Hz, H-1%), 3.54 (dd, 1 H, H-2), 4.25–4.18 (m,
2 H, H-5, H-6%a/b), 4.15–4.12 (m, 2 H, H-6a,
H-6b), 4.07–4.04 (m, 1 H, H-6%a, H-6%b), 3.95
(dd, 1 H, H-3), 3.85 (m, 1 H, H-5), 2.18–1.95
(7s, 21 H, CH3CO); 13C NMR (CDCl3): l
170.68–169.72(CꢁO), 134.12, 133.24. 133.21,
128.64, 127.90, 127.81, 127.68, 126.65, 126.55,
126.06 (10 C, ArC), 100.51 (C-1%), 97.24 (C-1),
72.76 (C-3), 70.81 (C-5/C-3%), 70.72 (C-5/C-3%),
70.33 (CH2), 68.79 (C-4), 68.49 (C-2%), 67.60
(C-5), 66.69 (C-4%), 62.67 (C-6%), 61.09 (C-6),
48.77 (C-2), 23.23 (CH3CON), 20.68–20.41 (6
C, CH3COO). Anal. Calcd for C37H45NO17:
C, 57.29; H, 5.85; N, 1.81. Found: C, 57.02;
H, 6.01; N, 1.61.
copyranosyl-(1“3)-2-azido-2-deoxy-a-
D
-gal-
actopyranoside (22) (0.093 g, 88%); Rf 0.32
(solvent B); [h]D +4° (c 0.2, CHCl3); H
1
NMR (CDCl3): Table 3; Anal. Calcd for
C31H38N4O13: C, 55.19; H, 5.68; N, 8.30.
Found: C, 54.95; H, 6.01; N, 8.25.
Compound 22 (0.1 g, 0.1 mmol) in MeOH
(5 mL) was treated with 20 wt.% palladium
hydroxide–charcoal (0.03 g) and hydrazine
(0.1 mL) under refluxing conditions for 2 h.
The solvent was evaporated to dryness, and
the residue was per-O-acetylated using 1:1
pyridine–Ac2O. The solvent was concd to dry-
ness, and the residue was purified by column
chromatography (EtOAc in hexane 50“66“
75%) to yield the fully acetylated target disac-
charide, 2-naphthylmethyl 2-acetamido-3,4,6-
2-Naphthylmethyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-i-
D
-glucopyranosyl-(1“3)-2-
-galac-
acetamido-4,6-di-O-acetyl-2-deoxy-h-
D
tri - O - acetyl - 2 - deoxy - b -
(1“3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-
a- -galactopyranoside (23) (0.055 g, 70%); Rf
D
- glucopyranosyl-
topyranoside (23).—A 1:1 mixture of ni-
tromethane and benzene (40 mL) was distilled
off until one-third of the solvent was removed
from the reaction flask. The mixture was then
cooled down to 40 °C and molecular sieves (4
D
1
0.57 (solvent C); [h]D +8° (c 0.2, CHCl3); H
NMR (CDCl3): l 7.88–7.43 (m, 7 H, ArH),
6.04 (d, 1 H, J2,NH 8.5 Hz, N-H), 5.80 (d, 1 H,
J2,NH 8.0 Hz, N-H), 5.39 (dd, 1 H, H-4), 5.35
(dd, 1 H, H-3%), 5.09 (d, 1 H, J1,2 3.5 Hz, H-1),
5.04 (dd, 1 H, H-4%), 4.96 (d, 1 H, J1%,2% 8.5 Hz,
H-1%), 4.85 (d, 1 H, CH2), 4.66 (d, 1 H, CH2),
4.46–4.42 (m, 1 H, H-2), 4.33–4.30 (m, 1 H,
H-6%a/b), 4.23–4.21 (m, 1 H, H-5), 4.17–4.14
(m, 1 H, H-6a/b), 4.09–4.04 (m, 2 H, H-6%a/b,
H-6a/b), 3.98 (dd, 1 H, H-3), 3.68–3.64 (m, 1
,
A, 4 g) were added followed, by mercuric
cyanide (0.3 g, 1.2 mmol) and mercuric bro-
mide (0.4 g, 1.1 mmol) with stirring. Com-
pound 17 (0.2 g, 0.5 mmol) was added, and
the reaction mixture was stirred for a further
30 min under argon. 2-Acetamido-3,4,6-tri-O-
acetyl-2-deoxy-a-
D
-glucopyranoside (0.4 g, 1.0
mmol) was added, and stirring was continued

298
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
column chromatography (19:1 CHCl3–
MeOH) to give 2-naphthylmethyl 2-aceta-
H, H-5%), 3.58–3.52 (m, 1 H, H-2%), 2.12–1.91
(7s, 21 H, CH3CO). 13C NMR (CDCl3):
171.13–169.36 (CꢁO), 134.03, 133.15, 128.55,
127.84, 127.75, 127.53, 126.53 (2 C), 126.42,
125.95 (10 C, ArC), 99.05 (C-1%), 96.97 (C-1),
72.46 (C-3), 71.97 (C-3%, C-5%), 70.18 (CH2),
68.83 (C-4), 68.31 (C-4%), 67.44 (C-5), 62.52
(C-6), 61.28 (C-6%), 55.22 (C-2%), 48.95 (C-2),
23.29 (CH3CON), 20.69 (6 C, CH3COO).
Anal. Calcd for C37H46N2O16: C, 57.28; H,
5.98; N, 3.62. Found: C, 56.99; H, 6.15; N,
3.72.
mido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-glucopy-
-galacto-
ranosyl-(1“6)-2-azido-2-deoxy-a-
D
pyranoside (26) (0.062 g, 82%); Rf 0.52 (sol-
vent B); [h]D +12° (c 0.2, CHCl3); H NMR
1
(CDCl3): Table 3; Anal. Calcd for C31H38-
N4O13: C, 55.19; H, 5.68; N, 8.30. Found: C,
54.89; H, 5.72; N, 8.30.
Compound 26 (0.05 g, 0.07 mmol) was
treated with hydrazine (0.02 g) and 20 wt.%
Pd–C (0.015 g), followed by acetylation with
1:1 pyridine–Ac2O to give the fully acetylated
target disaccharide, 2-naphthylmethyl 2-acet-
2-Naphthylmethyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-i-
D
-glucopyranosyl-(1“6)-2-
-galac-
acetamido-3,4-di-O-acetyl-2-deoxy-h-
D
amido-3,4,6-tri-O-acetyl-2-deoxy-b-
D
-gluco-
topyranoside (27).—To a suspension of com-
pound 16 (0.1 g, 0.3 mmol) in 2,2-
dimethoxypropane (5 mL) was added 10-cam-
phorsulfonic acid (0.04 g, 0.17 mmol), and the
mixture was stirred for 24 h at rt. Triethyl-
amine (0.1 mL) was added to quench the
reaction, and the mixture was concd to dry-
ness. The residue was treated with 9:1
MeOH–water (10 mL) and heated for 24 h
under gentle reflux. The reaction mixture was
again concd to dryness, and the residue was
purified by column chromatography (1:2
EtOAc–hexane) to give 2-naphthylmethyl 2-
pyranosyl-(1“6)-2-acetamido-3,4-di-O-ace-
tyl-2-deoxy-a- -galactopyranoside (27) (0.041
D
g, 71%). Rf 0.52 (solvent C), [h]D +8° (c 0.15,
1
CHCl3); H NMR (CDCl3): l 7.88–7.45 (m, 7
H, ArH), 5.61 (d, 1 H, J2,NH 9.5 Hz, NH),
5.50 (d, 1 H, J2,NH 8.5 Hz, NH), 5.37 (dd, 1 H,
H-4), 5.33 (dd, 1 H, H-3%), 5.18 (dd, 1 H, H-3),
5.03 (dd, 1 H, H-4%), 5.00 (d, 1 H, J1,2 3.5 Hz,
H-1), 4.88 (d, 1 H, CH2), 4.72 (d, 1 H, J1%,2%
8
Hz, H-1%), 4.66 (d, 1 H, CH2), 4.58 (m, 1 H,
H-2), 4.27–4.08 (m, 2 H, H-6%a/b, H-6a/b),
3.83 (m, 1 H, H-6a/b), 3.70 (dd, 1 H, H-2%),
3.64 (m, 1 H, H-5%), 3.55 (m, 1 H, H-5), 2.15,
2.07, 2.03, 2.02, 1.98, 1.91, 1.90 (7s, 21 H,
CH3CO); 13C NMR (CDCl3): l 170.88–168.32
(CꢁO), 134.14, 133.16, 133.06, 128.49, 127.93,
127.73, 127.28, 126.46, 126.33, 125.90 (10 C,
ArC), 100.37 (C-1%), 99.91 (C-1), 71.89 (C-5%),
71.56 (C-3%), 70.05 (CH2), 68.52 (2 C), 67.85,
67.64, 67.45, 61.90 (C-6%), 55.07 (C-2%), 47.80
(C-2), 23.90 (CH3CON), 20.72 (6 C,
CH3COO). Anal. Calcd for C37H46N2O16: C,
57.36; H, 5.98; N, 3.62. Found: C, 57.16; H,
6.21; N, 3.67.
azido-2-deoxy-3,4-O-isopropylidene-a- -gal-
D
actopyranoside (24) (0.096 g, 86%); Rf 0.62
1
(solvent B), [h]D +20° (c 0.2, CHCl3); H
NMR (CDCl3): Table 3; Anal. Calcd for
C20H23N3O5: C, 62.33; H, 6.01; N, 10.90.
Found: C, 61.88; H, 6.45; N, 11.02.
Compound 24 (0.09 g, 0.234 mmol) was
reacted with 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-a- -glucopyranoside (0.16 g, 0.44
D
mmol) in a similar manner as previously de-
scribed for compound 21 to give 2-naphthyl-
methyl
oxy-b-
2-acetamido-3,4,6-tri-O-acetyl-2-de-
-glucopyranosyl-(1“6)-2-azido-2-de-
Cell culture.—U937 human histiocytic
lymphoma cells were from the American Type
Culture Collection (CRL 1593.2) and were
grown in RPMI 1640 medium (GIBCO) sup-
plemented with 10% (v/v) fetal bovine serum
(Hyclone), glutamine (0.3 g/L), streptomycin
sulfate (100 mg/mL), and penicillin (100 Units/
mL) [27,28]. Human umbilical vein endothe-
lial cells (HUVEC) were from Clonetics
Corporation (CC-2519) and were grown in
Medium 199 (GIBCO) containing 20% (v/v)
fetal bovine serum, heparin (100 mg/mL,
D
oxy-3,4-O-isopropylidene-a- -galactopyrano-
D
side (25) (0.087 g, 52%); Rf 0.62 (solvent B),
1
[h]D +10° (c 0.2, CHCl3); H NMR data
(CDCl3): Table 3; Anal. Calcd for C34H42-
N4O13: C, 57.14; H, 5.92; N, 7.84. Found: C,
57.21; H, 6.02; 7.82.
Compound 25 (0.08 g, 0.11 mmol) was
treated with 70% AcOH (5 mL) at 80 °C for 2
h, after which time the solvent was concd to
dryness and the residue was subjected to

A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
299
washed with 100% MeOH, water, and 0.5 M
NaCl. The columns were washed sequentially
with 0.5 M NaCl (2.5 mL) and water (25 mL).
Radioactive oligosaccharides were eluted with
40% MeOH in water (2.5 mL), dried and
counted by liquid scintillation.
Sigma) and endothelial cell growth supple-
ment (100 mg/mL, Collaborative Biomedical).
All cell lines were maintained at 37 °C in a
humidified incubator containing 5% CO2 and
95% air.
Adhesion assays.—E-selectin expression on
human umbilical vein endothelial cells (HU-
VEC) was stimulated with 20 ng/mL TNF-a
(R & D Systems) [29]. After 5 h, the monolay-
ers were washed three times with Minimal
Essential Medium (MEM, GIBCO) and used
for adhesion assays. U937 cells (2×105/well)
in RPMI 1640 medium were incubated with
and without acetylated disaccharide and [3H-
methyl]thymidine (1 mCi/mL) for 2 days.
Washed cells in 250 mL of PBS (containing
Ca2+ and Mg2+) were then added to TNF-a
activated HUVEC and incubated for 35 min
at 4 °C. The plates were washed gently three
times with 1 mL of cold medium and the cells
were solubilized with 0.1 M NaOH. Radioac-
tivity was measured after neutralizing the soln
with AcOH. The values were normalized to
the radiospecific activity of the cells deter-
mined on a separate aliquot. Cell adhesion
was \80% dependent on E-selectin under
these conditions based on the inhibitory activ-
ity of anti-ELAM-1 mAb (R&D Systems).
Labeling studies.—The per-O-acetylated
disaccharides were dissolved in Me2SO and
added to the growth media to achieve the
concentrations indicated in Tables 1 and 2
and Fig. 2. For the labeling studies, cells were
incubated with 10 mCi/mL [6-3H]fucose (83
Ci/mmol), 10 mCi/mL [6-3H]galactose (32 Ci/
mmol), 10 mCi/mL [6-3H]glucosamine (33.3
Ci/mmol) or 1 mCi/mL [3H-methyl]thymidine
(83 Ci/mmol). After ꢀ24 h, the cells were
sedimented by centrifugation, and the amount
of radioactivity that was incorporated into
cellular glycoproteins was determined by pre-
cipitation with 10% trichloroAcOH (TCA).
After centrifugation, the pellets were washed
with 2% TCA, dissolved in 0.1 M NaOH, and
counted by liquid scintillation spectrometry
using UltimaGold (Packard) cocktail.
Acknowledgements
This work was supported by grant CA46462
(J.D.E.) from the National Cancer Institute.
We thank Herman Van Halbeek and the Gly-
cotechnology Core of the Glycobiology Re-
search and Training Center at UCSD for help
with NMR spectroscopy.
References
[1] D. Vestweber, J.E. Blanks, Physiol. Re6., 79 (1999)
181–213.
[2] T. Krause, G.A. Turner, Clin. Exp. Metastasis, 17 (1999)
183–192.
[3] Y.J. Kim, L. Borsig, H.L. Han, N.M. Varki, A. Varki,
Am. J. Pathol., 155 (1999) 461–472.
[4] H. Repo, J.M. Harlan, Ann. Med., 31 (1999) 156–165.
[5] A. Varki, J. Clin. In6est., 99 (1997) 158–162.
[6] A.K. Sarkar, K.S. Rostand, R.K. Jain, K.L. Matta, J.D.
Esko, J. Biol. Chem., 272 (1997) 25608–25616.
[7] A.K. Sarkar, T.A. Fritz, W.H. Taylor, J.D. Esko, Proc.
Natl. Acad. Sci. USA, 92 (1995) 3323–3327.
[8] D. Horton, Methods Carbohydr. Chem., 6 (1963) 282–
285.
[9] R.I. El-Sokkary, B.A. Silwanis, M.A. Nashed, H.
Paulsen, Carbohydr. Res., 203 (1990) 319–323.
[10] B. Helferich, W. Ost, Chem. Ber., 95 (1962) 2616–2622.
[11] W. Koenigs, E. Knorr, Ber. Dtsch. Chem. Ges., 34 (1901)
957–981.
[12] G.H. Veeneman, S.H. van Leeuwen, J.H. Van Boom,
Tetrahedron Lett., 31 (1990) 1331–1334.
[13] R.R. Schmidt, Angew. Chem., Int. Ed. Engl., 25 (1986)
212–235.
[14] H. Paulsen, K.-M. Steiger, Carbohydr. Res., 169 (1987)
105–125.
[15] J.O. Kihlberg, D.A. Leigh, D.R. Bundle, J. Org. Chem.,
55 (1990) 2860–2863.
[16] B. Helferich, J. Zirner, Chem. Ber., 95 (1962) 2604–
2611.
[17] G.J.F. Chittendon, J.G. Buchanan, Carbohydr. Res., 11
(1969) 379–385.
[18] R.R. Schmidt, W. Kinzy, Ad6. Carbohydr. Chem.
Biochem., 50 (1994) 21–124.
[19] R.U. Lemieux, R.M. Ratcliffe, Can. J. Chem., 57 (1979)
1244–1251.
To measure priming of oligosaccharides, the
conditioned medium was adjusted to 0.5 M
NaCl and centrifuged. The supernatants were
then applied to 0.2 mL Sep-Pak Vac RC C18
Cartridges (Waters), which had been pre-
[20] G. Grundler, R.R. Schmidt, Justus Liebigs Ann. Chem.,
(1984) 1826–1843.
[21] P.H. Amvam-Zollo, P. Sinay
9 , Carbohydr. Res., 150
(1986) 199–212.
[22] A.A. Malik, S.B. Preston, T.G. Archibald, M.P. Choen,
K. Baum, Synthesis, (1989) 450–451.

300
A.K. Sarkar et al. / Carbohydrate Research 329 (2000) 287–300
[23] D. Zhuang, A. Grey, M. Harris-Brandts, E. Higgins,
M.A. Kashem, J.W. Dennis, Glycobiology, 1 (1991)
425–433.
[24] M.F.A. Bierhuizen, M. Fukuda, Proc. Natl. Acad. Sci.
USA, 89 (1992) 9326–9330.
Biochem. Biophys. Res. Commun., 182 (1992) 1288–1295.
[27] S.J. Collins, R.C. Gallo, R.E. Gallagher, Nature, 270
(1977) 347–349.
[28] C. Sundstrom, K. Nilsson, Int. J. Cancer, 17 (1976)
565–577.
[25] S.F. Kuan, J.C. Byrd, C. Basbaum, Y.S. Kim, J. Biol.
Chem., 264 (1989) 19271–19277.
[26] N. Kojima, K. Handa, W. Newman, S. Hakomori,
[29] M.P. Bevilacqua, J.S. Pober, D.L. Mendrick, R.S. Co-
tran, M.A. Gimbrone Jr., Proc. Natl. Acad. Sci. USA, 84
(1987) 9238–9242.
.