Synthesis and molecular recognition of carbohydrate-centered multivalent glycoclusters by a plant lectin RCA120

Article abstract of DOI:10.1016/j.bmc.2005.06.036

Water soluble and lectin-recognizable carbohydrate-centered glycoclusters were prepared efficiently by the Huisgen 1,3-cycloaddition reaction of methyl-2,3,4,6-tetra-O-propargyl β-d-galactopyranoside with 2-azidoethyl glycosides of lactose and N-acetyllac

Full text of DOI:10.1016/j.bmc.2005.06.036

Bioorganic & Medicinal Chemistry 13 (2005) 6151–6157
Synthesis and molecular recognition of
carbohydrate-centered multivalent glycoclusters by
a plant lectin RCA120
Yongjun Gao,a Atsuko Eguchi,b Kazuaki Kakehib and Yuan C. Leea,*
aDepartment of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
bFaculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502, Japan
Received 11 May 2005; revised 13 June 2005; accepted 14 June 2005
Available online 27 July 2005
Abstract—Water soluble and lectin-recognizable carbohydrate-centered glycoclusters were prepared efficiently by the Huisgen
1,3-cycloaddition reaction of methyl-2,3,4,6-tetra-O-propargyl b-D-galactopyranoside with 2-azidoethyl glycosides of lactose and
N-acetyllactosamine. Their binding by a plant lectin RCA120 was examined by capillary affinity electrophoresis using fluores-
cence-labeled asialoglycans from human a1-acid glycoprotein. The glycoclusters showed 400-fold stronger inhibitory effect than free
lactose, manifesting strong multivalency effect.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
neutrophils to endothelial surfaces,13 neutralization of
viruses and toxins,14 and carbohydrate-based anticancer
therapy,15 despite some drawbacks.16 However, most
conventional methods for the preparation of glycoclus-
ters require lengthy and reiterative synthetic steps,
requiring protection and deprotection before and after
the glycocluster construction.
Carbohydrate–protein recognition is important in many
biological processes in living systems, such as signal
transductions, cell adhesion, inflammation, and cancer
metastasis, bacterial and viral infection.1–3 However, in
most cases, monosaccharide–protein interactions are
very weak (the Kd value being about 10ꢀ3–10ꢀ6 M). In
nature, the weak binding of monosaccharides is com-
pensated by multiple and simultaneous binding of many
sugar residues,4 a phenomenon now often referred to as
Ôglycoside clustering effectÕ.5,6 To investigate the carbo-
hydrate recognition with the consideration of glycoside
clustering effect, synthetic multivalent glycoconjugates
have been used extensively as mimetics of naturally
occurring branched carbohydrate ligands. Syntheses of
such compounds typically utilize polyfunctional mole-
cules as ÔscaffoldÕ to organize structurally well-defined
mono- or oligosaccharide units to fit conformation of
sugar-binding proteins. For example, monovalent sug-
ars were coupled to such scaffolds as cyclodextrins
(CDs),7 calix[4]-allenes,8 silsesquioxanes,9,10 and pep-
tides.11,12 Applications of the multivalent glycoconju-
gates include prevention of early adhesion of
To obtain simpler and more efficient glycoclusters, we
recently reported a new strategy based on the use of
thiol radical addition of unprotected x-thioglycosides
to octavinyloligosilsequioxane, eliminating the need to
protect and deprotect during glycoclusters construc-
tion.10 In recent years, the Huisgen 1,3-dipolar cycload-
dition reaction between an azide and terminal alkyne
has been gaining considerable attention as a powerful
approach for preparation of peptidotriazoles,17 fabrica-
tion of carbohydrate arrays,18 synthesis of multivalent
neoglycoconjugates,19 as well as labeling proteins and
cell surfaces.20
Carbohydrate molecules are innately polyfunctional,
and have been widely exploited as multivalent scaffolds
for the synthesis of oligosaccharide mimetics with out-
standing biological functions. For example, Starfish, a
powerful ligand for Shiga-like toxin was developed from
a glucoside derivative.14 We thought that the 1,3-dipolar
cycloaddition could be useful for preparation of carbo-
hydrate-centered glycoclusters by the simultaneous
Keywords: Lectin RCA120; 1,3-Cycloaddition reaction; Glycocluster;
Molecular recognition.
*
Corresponding author. Tel.: +1 410 516 7322; fax: +1 410 516
8716; e-mail: yclee@jhu.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.036

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Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
Figure 1. The outline of the synthesis of carbohydrate-centered glycoclusters.
assembly of alkynyl glycoside and azide glycosides. We
now describe in this paper, the first synthesis of meth-
yl-2,3,4,6-tetra-O-propargyl-b-D-galactopyranoside and
its use as a polyfunctional scaffold for construction of
glycocluster. Our facile synthesis involves rapid assem-
bling of multivalent glycoclusters in a convergent way
from unprotected lactose and N-acetyllactosamine
derivative using the Huisgen 1,3-cycloaddition reaction
as the key step. Our strategy is summarized in Figure 1.
types of normal and pathological reactions. In addition,
lactosyl group is the primer disaccharide in the biosyn-
thesis of most glycosphingolipids. LacNAc is also a
precursor of sialyl Lewisx, which plays an important role
in selectin-mediated adhesion of neutrophils to endothe-
lial cells, the initial event in many inflammatory respons-
es.25,26 Thus, we chose azide-bearing glycosides of
lactose and N-acetyllactosamine as monomeric struc-
tures for clustering.
Efficacy of the synthesized glycoclusters was evaluated
by capillary affinity electrophoresis (CAE).21 Inhibition
of the binding of the desialylated N-linked oligosaccha-
rides from a1-acid orosomucoid (AGP-asialoglycans)
to RCA120 by the glycoclusters was quantified and
compared with that by lactose.
Although compound 5 has been synthesized before,27
we used a different route for synthesis as shown in
Scheme 2. Per-O-benzoylated lactosyl bromide 228
was reacted with 2-chloroethanol under the catalysis
of silver triflate (AgOTf) to give the b-lactoside deriva-
tive 3 in near quantitative yield. The desired b-ano-
meric configuration was confirmed by the relatively
large coupling constant of the anomeric proton signal
(J1,2 = 8.0 Hz). It should be noted that Bz-protected
lactosyl bromide was chosen over the conventional
acetylated counterpart to exclude the possibility of
orthoester or a-glycoside formation.29 The chloroethyl
glycoside was then reacted with sodium azide (NaN3)
via an SN2 type reaction to give 2-azidoethyl glycoside
4 in high yield (90%). The two-step procedure was used
instead of direct glycosylation of 2-azidoethanol which
is potentially explosive.30 Subsequent deprotection
2. Results and discussion
2.1. Synthesis of the tetravalent carbohydrate scaffold
Because of their easy availability, biocompatibility, low
toxicity, polyfunctionality, and intrinsic chirality, carbo-
hydrate molecules have been widely used as cores for
synthesis of a variety of glycoclusters and glycodendri-
mes.22–24 In our current study, we chose the inexpensive
commercially available methyl b-D-galactopyranoside as
the core molecule. To transform methyl b-D-galactopy-
ranoside into a suitable template for multivalent glyco-
cluster assembly, uniform propargylation of all four
hydroxyl groups of methyl b-D-galactopyranoside was
accomplished with propargyl bromide and NaH in
DMF to give the alkyne cluster 1 in 80% yield (Scheme
1). The product was purified by standard silica gel
chromatography.
´
under the Zemplen condition gave the unprotected
glycoside 5.
The synthesis of the x-azido-glycoside of N-acetyllactos-
amine, 9,31 is shown in Scheme 2. Per-O-acetylation of
starting material, N-acetyllactosamine, produced com-
pound 632 in quantitative yield. This was converted into
the oxazoline derivative 7 by treating with TMSOTf.33
The oxazoline 7 was then allowed to react with 2-azido-
ethanol using pyridinium p-toluenesulfonate (PPTS) as a
promoter,34 giving the corresponding b-glycoside 8.35
2.2. Synthesis of azide glycosides
´
Subsequent deprotection under the Zemplen condition
Lactose (Lac) and N-acetyllactosamine (LacNAc) are
two of the most common structures in a variety of gly-
coconjugates, and are known to be involved in various
gave the unprotected glycoside 9.31
2.3. Synthesis of tetravalent carbohydrate-centered
glycoclusters
Several neoglycoconjugates have been prepared by
copper (I)-catalyzed 1,3-cycloaddition reaction through
the use of protected glycosides in organic solvents.19
We show that unprotected glycoside can be used in this
reaction equally efficiently, and this reduces the number
of protection and deprotection steps before and after
glycocluster assembly. Moreover, our method uses
Scheme 1. Synthesis of carbohydrate scaffold.

Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
6153
Scheme 2. Synthesis of 2-azidoethyl glycosides.
aqueous solution and thus is more compatible with envi-
ronmental protection.
2.4. Inhibition of RCA120 binding of asialoglycans from
human al-acid glycoprotein by the glycoclusters
As shown in Scheme 3, direct assembly of glycoclusters
was achieved by reacting alkynyl galactoside and unpro-
tected azide glycosides in the presence of CuSO4/sodium
ascorbate in water36 to give the corresponding products
10 and 11, which are easily isolated in high yields by
simple gel filtration. They were fully characterized by 1H
NMR, 13C NMR, and MALDI-TOF mass spectroscopy.
RCA120 is a bivalent lectin specifically recognizing the
exposed b-D-galactose residues. In solution, RCA120
binds two galactosides, one by each subunit.37,38 The
binding affinity of glycoclusters 10 and 11 to RCA120
was estimated by capillary affinity electrophoresis
(CAE) by inhibition of the binding of asialoglycans
from human a1-acid glycoprotein (AGP-asialoglycans).
AGP contains di-, tri-, and tetra-antennary carbohy-
drate chains, and the asialoglycans thereof have termi-
nal Galb1-4GlcNAc on all branches and show high
affinity toward RCA120. AGP-asialoglycans showed
discrete peaks (A1–A5), identified previously,39 as
shown in Figure 2.
Scheme 3. Assembly of glycoclusters.
Figure 2. Capillary electrophoresis of AGP-asialoglycans.

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Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
In the presence of RCA120, AGP-asialoglycans were
bound by RCA120 and their peaks were broadened as
shown in Figure 3. All peaks broadened as a function
of RCA120 concentration, but the triantennary glycan
(A3) and tetra-antennary glycans (A4 and A5) were most
2 and 3 (top profile). For comparison, free lactose was
used as monovalent reference under the same experi-
mental conditions. The minimum concentrations of
saccharide units to completely inhibit the binding
between the AGP-asialoglycans and RCA120 (ICmin
are summarized in Table 1.
)
sensitive to lectin RCA120
.
The peak broadening was increasingly suppressed when
the increasing amounts of the glycocluster were added to
the sample. Eventually the peaks became as discrete as
the free (unbound) oligosaccharides as shown in Figures
These data indicate that glycoclusters 10 and 11 are equal-
ly well recognized by RCA120 and they are excellent
inhibitors for the binding of lectin RCA120 to AGP-asi-
aloglycans. Their inhibitory power is much stronger than
the free lactose (minimum inhibition concentration of
20 mM) by a factor of 400 (minimum inhibition concen-
tration of 50 lM). From these inhibition experiments, it
appears that a significant effect of multivalency is
observed for the glycoclusters. A typical inhibitory
experiment of glycocluster is illustrated in Figure 4.
3. Conclusion
A simple and effective preparation of carbohydrate-cen-
tered glycoclusters based on 1,3-cycloaddition reaction
of uniformly alkynylated carbohydrate core with
unprotected x-azido-glycosides was developed. We
demonstrated the usefulness of this approach by the
preparation glycoclusters of lactose and N-acetyllactos-
amine, which were shown to be bound to RCA120 400-
fold more strongly than monovalent lactose. Given the
modular nature of this strategy, this approach may offer
an alternative avenue for preparing glycoclusters with
different densities and spacers, expanding the repertoire
of the existing glycoclusters for bioassays and
biorecognition.
Figure 3. Capillary affinity electrophoresis of AGP-asialoglycans in
the presence of various concentrations of RCA120.
Table 1. Inhibition of the binding between RCA120 and AGP-
asialoglycans by the glycoclusters and lactose
ICmin (M)b
a
Inhibitor
4. Experimental
Lactose
Glycocluster 10
Glycocluster 11
20 · 10ꢀ3
50 · 10ꢀ6
50 · 10ꢀ6
4.1. General procedures
a Minimum inhibition concentration.
b Molarity of saccharide unit.
Chemicals were from Aldrich (Milwaukee, WI) and used
as received. Column chromatography was carried out
Figure 4. Inhibition effects of glycocluster 10 on the binding between RCA120 and AGP-asialoglycans.

Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
6155
using E. Merck silica gel 60F (230–400 mesh). Analytical
thin-layer chromatography (TLC) was performed on
aluminum sheets coated with silica gel 60 F254
(0.25 mm thickness, E. Merck, Darmstadt, Germany).
Detection of UV-absorbing compounds on TLC plates
was by quenching of the silica gel-imbedded fluorescence
and/or charring with 15% H2SO4 in ethanol solution fol-
lowed by heating at ca. 180 ꢁC. The ratios of solvents
used for TLC are expressed as vol/vol. Gel filtration
chromatography was performed using a column packed
with Sephadex G-15 (2·145 cm) eluting with distilled
water. Carbohydrates were determined with a version
of the phenol–sulfuric acid method.40 Melting points
were determined with a Fisher-Johns apparatus and are
pearance of the starting material. The reaction mixture
was cooled to 0 ꢁC and MeOH (20 mL) was added.
The solvents were removed under reduced pressure.
The residue was suspended in water (30 mL) and
extracted with ethyl acetate (350 mL). The combined
organic layer was washed with satd aqueous NaCl
(50 mL), dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to give a brown
oil which was purified by silica gel chromatography
(hexanes/EtOAc 3:1) to give a pale yellow foam
(556 mg, 80%). Rf = 0.42 (hexanes/EtOAc 2:1). 1H
NMR (400 MHz, CDCl3, rt) d 4.33–4.49 (4 dd, 8H,
J = 14.8 and 2.4 Hz, 4-OCH2), 4.20–4.26 (m, 3H), 4.04
(br t, 1H), 3.80 (dd, 1H, J = 10 and 2.0 Hz), 3.71 (dd,
1H, J = 10 and 2.0 Hz), 3.58–3.61 (m, 3H), 3.52 (s,
3H, OCH3), 2.46 (m, 4H, 4-CH); 13C NMR
(100 MHz, CDCl3, rt): d 104.21, 80.79, 80.28, 80.09,
79.52, 79.30, 74.67, 74.49, 74.04, 73.32, 73.20, 68.94,
59.90, 59.51, 58.99, 58.71, 56.92; MALDI-TOF MS:
m/z calcd for C19H22NaO6 (M+Na), 369.1; found 368.9.
1
not corrected. H NMR and 13C NMR spectra were
recorded with a Varian-400 NMR spectrometer at nom-
inal resonance frequencies of 400 and 100 MHz, respec-
tively, in CDCl3 (referenced to internal Me4Si at dH
0 ppm, dC 0 ppm) and D2O (referenced to internal ace-
tone at dH 2.225 ppm, dc 29.8 ppm). The chemical shifts
(d) were expressed in parts per million (ppm). First order
J values were given in hertz. Fast atom bombardment
mass spectra (FAB MS) data were obtained using
m-nitrobenzyl alcohol as matrix. Matrix-assisted laser
desorption ionization time-of-flight mass spectra (MAL-
DI-TOF MS) were recorded with a MALDI-Compact
(Kratos) instrument in the positive mode using 2,5-
dihydroxybenzoic acid (DHB) as matrix unless otherwise
indicated and an average of 50 laser shots per sample.
4.4. 2-Chloroethyl (2,3,4,6-tetra-O-benzoyl-b-D-galacto-
pyranosyl)-(1!4)-2,3,6-tri-O-benzoyl-b-D-glucopyrano-
side (3)
A mixture of lactosyl bromide28 (2.64 g, 2.33 mmol) and
2-chloroethanol (4.0 mL, 59.6 mmol) in dry CH2C12
˚
(20 mL) was stirred under N2 with MS 4 A (3 g) for
1 h, then cooled to ꢀ15 ꢁC and powdered AgOTf
(1.3 g, 5.06 mmol) was added. The reaction was allowed
to stir at 0 ꢁC for 2 h until the complete disappearance of
the starting material, quenched by the addition of Et3N
(5 mL), filtered through a pad of Celite, diluted with
CH2Cl2 (100 mL), washed with satd aqueous NaHCO3
(50 mL), 2 M aqueous Na2S2O3, water (50 mL), dried
(Na2SO4), and evaporated to give a residue, which was
purified by silica gel chromatography (toluene/acetone
25:1) to afford the product as a white foam (2.49 g,
95%). Rf = 0.29 (toluene/acetone 20:1). 1H NMR
(400 MHz, CDCl3, rt) d 7.92–8.00 (m, 12H), 7.11–7.74
(m, 23H, arom), 5.79 (dd, 1H, J = 9.6 Hz, H-3, Glc),
5.68–5.73 (m, 2H), 5.45 (dd, 1H, J = 9.6 and 7.6 Hz,
H-2), 5.37 (dd, 1H, J = 10.4 and 3.6 Hz), 4.86 (d, 1H,
J = 8.0 Hz, H-1, Gal), 4.76 (d, 1H, J = 8.0 Hz, H-1,
Glc), 4.60 (dd, 1H, J = 12.0 Hz, H-6a, Glc), 4.48 (dd,
1H, J = 4.4 Hz, H-6b, Glc), 4.24 (t, 1H, J = 9.6 Hz,
H-4, Glc), 3.98, 3.75 (2 m, 2H, OCH2CH2N3), 3.84–
3.89 (m, 2H), 3.69–3.75 (m, 2H), 3.49 (m, 2H,
OCH2CH2N3); 13C NMR (100 MHz, CDCl3, rt): d
165.83, 165.57, 165.40, 165.38, 165.25, 165.22, 164.79,
133.56, 133.41, 133.28, 133.21, 129.99, 129.87, 129.74,
129.68, 129.64, 129.58, 129.53, 129.44, 129.39, 129.27,
128.82, 128.63, 128.59, 128.52, 128.34, 128.25, 101.34,
100.98, 75.95, 73.11, 72.72, 71.74, 71.53, 71.39, 69.85,
67.50, 62.26, 61.04, 42.18; MALDI-TOF MS: m/z calcd
for C63H53NaClO18 (M+Na), 1155.3; found 1156.1.
4.2. Procedures for capillary affinity electrophoresis (CAE)
Capillary affinity electrophoresis was performed with a P/
ACE MDQ glycoprotein system (Beckman Coulter, Ful-
lerton, CA, USA) equipped with an eCAP N-CHO capil-
lary (20 cm effective length, 30 cm total length, 50 lm i.d.,
Beckman Coulter) using an argon laser-induced fluores-
cence detector as described previously.21 Detection was
performed by installing a 520 nm filter for emission with
a 488 nm argon laser for excitation. Tris–acetate buffer
(100 mM, pH 7.4) was used as an electrolyte throughout
the work. The sample solution was introduced to the cap-
illary by pressure method (0.5 psi, 5 s). Separation was
performed at 25 ꢁC at the applied potential of 18 kV. Data
were collected and analyzed with a standard 32 Karat
software (Version 4.0, Beckman Coulter) on Microsoft
Windows 2000. The procedures are briefly as follows.
First, a mixture of fluorescence-labeled oligosaccharides
only was analyzed. Then, the capillary was filled with
the same electrolyte, but containing the lectin at a speci-
fied concentration, and the same mixture of fluorescent
oligosaccharides was analyzed.
4.3. Methyl-2,3,4,6-tetra-O-propargyl-b-D-galactopyran-
oside (1)
To a solution of methyl b-D-galactopyranoside (388 mg,
2 mmol) in dry DMF (10 mL) was added NaH (60%
w/w in mineral oil, 480 mg, 12 mmol) at 0 ꢁC with fre-
quent venting. After stirring for 20 min, propargyl bro-
mide (80% in toluene, 2.25 mL, 20 mmol) was added
slowly and the mixture was stirred at 0 ꢁC for 20 min
and maintained at rt for 12 h. TLC indicated the disap-
4.5. 2-Azidoethyl (2,3,4,6-tetra-O-benzoyl-b-D-galactopyr-
anosyl)-(1!4)-2,3,6-tri-O-benzoyl-b-D-glucopyranoside (4)
To a solution of the chloride 3 (2.15 g, 1.90 mmol) in dry
DMF (10 mL) were added NaN3 (1.23 g, 19 mmol) and
18-crown-6 (50 mg, 0.19 mmol). The resulting mixture

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Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
was heated at 70 ꢁC with stirring for 36 h. The solvent
was removed under reduced pressure, the residue was
dissolved in EtOAc (100 mL), and washed with brine
(50 mL) and water (50 mL). The organic layer was dried
over anhydrous Na2SO4 and concentrated under re-
duced pressure. The residue was subjected to silica gel
column chromatography with toluene–acetone (25:1)
as the eluent to give 4 (1.95 g, 90%). Rf = 0.28 (tolu-
ene/acetone 20:1). 1H NMR (400 MHz, CDCl3, rt) d
7.95–8.02 (m, 12H), 7.13–7.74 (m, 23H, arom), 5.82
(dd, 1H, J = 9.6 Hz, H-3, Glc), 5.71–5.76 (m, 2H), 5.49
(dd, 1H, J = 9.6 and 7.6 Hz, H-2), 5.40 (dd, 1H,
J = 10.4 and 3.6 Hz), 4.90 (d, 1H, J = 8.0 Hz, H-1,
Gal), 4.78 (d, 1H, J = 7.6 Hz, H-1, Glc), 4.63 (dd, 1H,
J = 11 Hz, H-6a, Glc), 4.49 (dd, 1H, J = 4.4 Hz, H-6b,
Glc), 4.28 (t, 1H, J = 9.6 Hz, H-4, Glc), 3.98, 3.75
(2 m, 2H, OCH2CH2N3), 3.85–3.93 (m, 2H), 3.63–3.75
(m, 3H), 3.38, 3.24 (2m, 2H, OCH2CH2N3); 13C NMR
(100 MHz, CDCl3, rt): d 165.84, 165.57, 165.40,
165.22, 165.19, 164.80, 133.56, 133.41, 133.28, 133.21,
133.18, 129.99, 129.80, 129.74, 129.68, 129.64, 129.57,
129.53, 129.45, 129.39, 129.31, 128.82, 128.63, 128.58,
128.52, 128.31, 128.25, 101.07, 100.98, 75.93, 73.11,
72.82, 71.75, 71.60, 71.40, 69.88, 68.48, 67.50, 62.22,
61.04, 50.53; MALDI-TOF MS: m/z calcd for
C63H53N3Na18 (M+Na), 1162.3; found 1162.6.
2-azidoethanol (1.28 mL, 16.9 mmol, 9.6 equiv) and
pyridinium p-toluenesulfonate (PPTS)34 (45 mg,
0.175 mmol). The mixture was stirred under a nitrogen
atmosphere for 2 h at 70 ꢁC. The solution was cooled,
neutralized with pyridine (3 mL), diluted with chloro-
form (50 mL), and stirred in ice-water. The organic
layer was washed successively with aqueous sodium
hydrogen carbonate (30 mL) and water (30 mL), dried
over anhydrous Na2SO4, and evaporated. The residual
syrup was purified by silica gel chromatography (hex-
anes/acetone 1:1) to give pure product 8 (880 mg,
1
72%). H NMR (400 MHz, CDCl3, rt) d 5.78 (d, 1H,
J = 9.2 Hz), 5.36 (d, 1H, J = 3.2 Hz), 5.12 (m, 2H),
4.98 (dd, 1H, J = 10.4 and 3.2 Hz), 4.58 (d, 1H,
J = 7.6 Hz), 4.51–4.55 (m, 2H), 4.09–4.16 (m, 4H),
3.97–4.07 (m, 3H), 3.89 (t, 1H, J = 7.0 Hz), 3.81 (t,
1H, J = 8.8 Hz), 3.67 (m, 2H), 3.48 (m, 1H), 3.26 (m,
1H), 2.18 (s, 3H, CH3CO), 2.16 (s, 3H, CH3CO),
2.12 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 2.06 (m,
6H, 2 CH3CO), 1.97 (s, 3H, CH3CO); 13C NMR
(100 MHz, CDCl3, rt)
d 170.64, 170.41, 170.14,
170.08, 169.36, 101.02, 100.82, 75.73, 72.75, 72.27,
70.82, 70.73, 69.08, 68.24, 66.60, 62.10, 60.78, 53.26,
50.62, 23.29, 20.89, 20.86, 20.66, 20.53; MALDI-TOF
MS: m/z calcd for C28H41N4NaO17 (M+Na), 727.2;
found 727.3.
4.6. 2-Azidoethyl (b-D-galactopyranosyl)-(l!4)-b-D-gluco-
4.8. 2-Azidoethyl(b-D-galactopyranosyl)-(1!4)-O-2-acet-
amido-2-deoxy-b-D-glucopyranoside (9)31
pyranoside (5)27
To a solution of 4 (2.28 g, 2 mmol) in dry methanol
(30 mL) was added NaOMe (50 mg). The mixture was
stirred for 16 h at room temperature. The solvent was
removed by evaporation. The residue was dissolved in
water (30 mL) and neutralized with Dowex 50Wx8
(H+), filtered, and extracted with toluene (2·30 mL)
and ethyl acetate (20 mL). The water layer was lyophi-
lized to give a powder (740 mg, 91%). 1H NMR
(400 MHz, D2O, rt) d 4.53 (d, 1H, J1,2 = 8.4 Hz, H-1),
4.43 (d, 1H, J = 8.0 Hz, H-10), 3.78, 4.05 (2m, 2H,
OCH2CH2N3), 3.91 (d, 1H), 3.71–3.86 (m, 5H), 3.65
(m, 3H), 3.60 (br, 1H), 3.55 (m, 3H), 3.34 (t, 1H); 13C
NMR (100 MHz, D2O, rt): d 102.49, 101.74, 77.88,
74.92, 74.38, 73.90, 72.34, 72.08, 70.51, 68.11, 60.59,
59.61, 50.10; MALDI-TOF MS: m/z calcd for
C14H25N3NaO11 (M+Na), 434.1; found 434.2.
To a solution of 8 (704 mg, 1 mmol) in dry methanol
(15 mL) was added NaOMe (30 mg). The mixture was
stirred for 3 h at room temperature. The solution was
neutralized with Dowex 50Wx8 (H+). After removing
the resin by filtration, the filtrate was concentrated un-
der reduced pressure to give a white powder (430 mg,
95%). 1H NMR (400 MHz, D2O, rt) d 4.60 (d, 1H,
J1,2 = 8.0 Hz), 4.47 ðd; 1H; J1 ;2 ¼ 8.0 Hz; H-10Þ, 3.97
(d, 1H, H-40), 3.78, 4.06 (2m, 2H, OCH2CH2N3),
3.80 (dd, 1H, H-2), 3.74 (dd, 1H, H-30), 3.72 (dd,
1H, H-4), 3.69 (dd, 1H, H-30), 3.55 (dd, 1H, H-20),
3.41–3.51 (2m, 2H, OCH2CH2N3), 2.03 (s, 3H,
CH3CO); 13C NMR (100 MHz, D2O, rt): d 174.21,
102.44, 100.53, 77.96, 74.92, 74.37, 72.05, 70.52,
68.33, 60.59, 59.58, 54.57, 49.91, 21.83; MALDI-
TOF MS: m/z calcd for C16H28N4NaO11 (M+Na),
475.1; found 475.0.
0
0
4.7. 2-Azidoethyl (2,3,4,6-tetra-O-acetyl-b-D-galactopyr-
anosyl)-(1!4)-O- 2-acetamido-3,6-di-O-acetyl-2-deoxy-
b-D-glucopyranoside (8)35
4.9. General procedures for synthesis of glycoclusters
Methyl-2,3,4,6-tetra-O-propargyl b-D-galactopyrano-
side 1 (0.06 mmol) and azide glycoside (0.48 mmol) were
suspended in water (12 mL). Sodium ascorbate
(0.044 mmol, 45 lL of freshly prepared 1 M solution
in water) was added, followed by copper (II) sulfate pen-
tahydrate (0.022 mmol, 22 lL of 1 M solution in water).
The mixture was stirred vigorously for 24 h, at which
point the mixture cleared and TLC analysis indicated
complete consumption of the reactants. The reaction
mixture was reduced to about 2 mL and applied to a
Sephadex G-15 column (2·145 cm) eluting with distilled
water. Appropriate fractions were pooled and lyophi-
lized to give white foam.
A solution of N-acetyllactosamine peracetate 632 (1.8 g,
2.64 mmol) in dry 1,2-dichloroethane (12 mL) was
treated with TMSOTf (0.5 mL, 2.63 mmol) under a
nitrogen atmosphere, and the mixture was stirred over-
night at 50 ꢁC. Triethylamine (1.8 mL) was added and
the solvent was evaporated. The resulting residue was
purified by silica gel chromatography, eluting with
toluene–ethyl acetate–triethylamine (100:200:1), to
give the syrupy oxazoline derivative 7 (1.3 g, 80%).
To a solution of oxazoline derivative 733 (1.08 g,
1.75 mmol) in 1,2-dichloroethane (35 mL) were added

Y. Gao et al. / Bioorg. Med. Chem. 13 (2005) 6151–6157
6157
1
4.9.1. Glycocluster 10. Yield, 83%; H NMR (400 MHz,
17. Tornoe, C. W.; Christensen, C.; Meldal, J. J. Org. Chem.
2002, 67, 3057.
D2O, rt) d 8.12 (m, 4H), 4.6 (m, 15H), 4.44 (m, 9H), 4.28
(m, 6H), 4.12 (m, 6H), 3.92 (m, 8H), 3.76–3.52 (m, 40H),
3.25 (m, 5H); 13C NMR (100 MHz, D2O, rt): d 143.48,
143.12, 125.35, 103.12, 102.50, 101.88, 77.87, 74.93,
74.33, 73.81, 72.19, 72.08, 70.51, 68.11, 67.74, 64.70,
62.93, 62.55, 60.59, 59.59, 56.96. 49.93. MALDI-TOF
MS: m/z calcd for C75H122N12NaO50 (M+Na), 2013.7;
found, 2014.2.
18. Fazio, F.; Bryn, M. C.; Blixt, O.; Paulson, J. C.; Wong,
C.-H. J. Am. Chem. Soc. 2002, 124, 14397.
19. Perez-Balderas, F.; Ortega-Munoz, M.; Morales-Sanfru-
tos, J.; Hernandez-Mateo, F.; Calvo-Flores, F. C.; Calvo-
Asin, J. A.; Isac-Garcia, J.; Santoyo-Gonzalez, F. Org.
Lett. 2003, 5, 1951.
20. Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem.
Soc. 2003, 125, 4686.
21. Nakajima, K.; Oda, Y.; Kinoshita, M.; Kakehi, K.
J. Proteome Res. 2003, 2, 81.
1
4.9.2. Glycocluster 11. Yield, 88%; H NMR (400 MHz,
22. Wong, C.-H.; Ye, X.-S.; Zhang, Z. J. Am. Chem. Soc.
1998, 120, 7137.
23. Wunberg, T.; Kallus, C.; Opatz, T.; Henke, S.; Schmidt,
W.; Kunz, H. Angew. Chem. Int. Ed. Engl. 1998, 37,
2503.
24. Dubber, M.; Lindhorst, T. K. Org. Lett. 2001, 3, 4019.
25. Fenderson, B. A.; Eddy, E. M.; Hakomori, S.-I. Bioessays
1990, 12, 173.
D2O, rt) d 8.11(m, 4H), 4.33 (m, 9H), 4.05 (m, 11H),
3.94 (m, 13H), 3.66–3.75 (m, 43H), 3.55 (m, 12H), 1.89
(br s, 12H); 13C NMR (100 MHz, D2O, rt): d 173.6,
140.03, 124.79, 102.80, 102.43, 100.44, 77.91, 74.91,
74.31, 72.06, 71.78, 70.51, 68.10, 67.03, 60.58, 59.56,
54.32, 49.97, 21.72. MALDI-TOF MS: m/z calcd for
C83H134N16O50 (M+H), 2156; found, 2156.
26. Lasky, L. A. Annu. Rev. Biochem. 1995, 64, 113.
27. Chernyak, A.; Oscarson, S.; Turek, D. Carbohydr. Res.
2000, 329, 309.
Acknowledgment
28. Lichtenthaler, F. W.; Kaji, E.; Weprek, S. J. Org. Chem.
1985, 50, 3505.
29. Garegg, P. J.; Norberg, T. Acta Chem. Scand. B 1979, 33,
116.
This work was supported by the National Institutes of
Health Research Grant No. RO1 DK 009700.
30. Yudina, O. N.; Sherman, A. A.; Nifantiev, N. E. Carbo-
hydr. Res. 2001, 332, 363.
31. Blixt, O.; Brown, J.; Schur, M. J.; Wakarchuk, W.;
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32. Lemieux, R. U.; Abbas, S. Z.; Burzynska, M. H.; Ratcliffe,
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