A convergent synthesis of core 2 branched sialylated and sulfated oligosaccharides

Article abstract of DOI:10.1016/S0968-0896(02)00246-8

A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.

Full text of DOI:10.1016/S0968-0896(02)00246-8

Bioorganic & Medicinal Chemistry 10 (2002) 3673–3684
A Convergent Synthesis of Core 2 Branched Sialylated and
Sulfated Oligosaccharides
Jie Xia, James L. Alderfer, Thamarapu Srikrishnan,
E. V. Chandrasekaran and Khushi L. Matta*
Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets,
Buffalo, NY 14263, USA
Received 7 January 2002; accepted 12 April 2002
Abstract—A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and
sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete
regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary
hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor
10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1–3 were then obtained by
the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1–3 obtained, biological evaluations
of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
found to be a part of certain sulfated mucins from colon
cancer.6 We have examined the specificity of sulfo-
transferases present in human breast and colon tissues
and have made some very important observations. Ours
was the first report wherein a well-defined core 2 tetra-
saccharide structure was used to distinguish the sulfo-
transferase activities in these different human tissues.7
Two distinct types of Gal:3-O-sulfotransferases were
revealed. One was highly specific for the Galb1!
4GlcNAc branch of this tetrasaccharide and the other
showed preference for the Galb1!3GalNAc arm. We
have also examined 3-O-sulfotransferase activities7b in
various human tumor cell lines, for example colon
LS180, SW1116 and breast line 435/LCC6. In fact, the
availability of the modified analogues A and B, shown
in Figure 1, enabled us to distinguish and therefore
examine the status of these two different sulfo-
transferases in these sources.
Great advancement in synthetic carbohydrate chemistry
has been realized.1,2 The complex oligosaccharide
molecules resulting from these efforts can provide for a
wealth of information and offer insights into important
biological processes.3 The natural mucin ligands for cell
adhesion proteins and various tumor-associated
O-linked glycoproteins are known to contain the
Galb1!4GlcNAcb1!6(Galb1!3) GalNAca sequence
as their inner core structure. However, relatively little is
known about the precise substrate specificities of the
enzymes that can further modify this tetrasaccharide
moiety. From structural studies conducted on natural
ligands for selectins (such as GlyCAM-1),4 it has been
shown that the sulfate ester (designated by SE- in for-
mulae) is generally located at the C-6 position of
GlcNAc or galactose, whereas, sulfate is present at the
C-3 position of galactose in sulfated mucin from colon
cancer.5 Interestingly, the NeuAca2!3(SE-6Gal)b1!
4GlcNAc sequence occurs as a part of GlyCAM-1,
whereas, NeuAca2!3(SE-6Gal)b1!3GalNAc has been
We hereby describe the synthesis of modified penta-
saccharides 1 and 2 as potential acceptors for 6-O-sulfo-
transferase capable of synthesizing the NeuAca2!
3(SE-6Gal)b1!3GalNAc and NeuAca2!3(SE-6Gal)
b1!4GlcNAc sequences, respectively. We argue that
these branched compounds will be useful as preferred
acceptors even in the presence of 3-O-sulfotransferase
*Corresponding author. Tel.: +1-716-845-2300; fax: +1-716-845-
3458; e-mail: khushi.matta@roswellpark.org
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00246-8

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J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
Figure 1.
capable of acting upon the Galb1!4GlcNAc or
Galb1!3GalNAc sequences of O-linked glycoproteins.
We also describe the synthesis of a sialylated and
position of GlcNphth was designed to enable the use of
methyl-tri-O-benzyl-1-thio-b-l-fucoside 12 as a donor.
The 2-naphthylmethyl (NAP) group was recently intro-
duced for hydroxyl protection and this group is stable
under a variety of strong Lewis acid and base condi-
tions, or even in inorganic acid and base conditions. The
most important factor is that the NAP group can be
selectively removed in the presence of benzyl groups by
DDQ.9 Therefore, the complete syntheses of targets 1–3
are highly dependent upon the construction of dis-
accharide donor 4, trisaccharide acceptor 5, trisacchar-
ide donor 6, disaccharide acceptor 7, sialylated Lewisx
tetrasaccharide donor 9 and disaccharide acceptor 8.
The novel sialyl Le donor 9 and disaccharide acceptor
8 were prepared according to our previously reported
protocol.8e Preparations of the key intermediate build-
ing blocks 4, 5 and 7 are illustrated in Schemes 3–5.
sulfated
hexasaccharide,
NeuAca2!3Galb1!4
[Fuca1!3]SE-6GlcNAcb1!6(Galb1!3)GalNAca1!
OMe 3, which is a part of GlyCAM-1, one of the
naturally occurring ligands for 1-selectin.
Results and Discussion
Our strategies for the assembly of oligosaccharides 1, 2
and 3 is based on the observation of the much higher
reactivity of a primary hydroxyl group over a secondary
axial hydroxyl group in glycosylation reactions.8aÀe This
fact provides for a general route to the construction of
core 2 branched oligosaccharide structures and their
analogues. Therefore, pentasaccharides 1 and 2 were,
respectively, synthesized from the MeO-3Galb(1!4)
GlcNphth disaccharide fragment 4 and the Neu5A-
ca(2!3)Galb(1!3)GalNAc trisaccharide fragment 5,
and the Neu5Aca(2!3)Galb(1!4)GlcNphth tri-
saccharide donor 6 with MeO-3Galb(1!3)GalNAc dis-
accharide acceptor 7. Similarly, hexasaccharide 3 was
constructed from the Galb(1!3)GalNAc fragment 8
and the sialylated Lewis donor 9 (Scheme 1). The key
monosaccharide building blocks, 10–18, required for the
construction of these advanced intermediates are illu-
strated in Scheme 2.
Construction of target 1 is shown in Schemes 3 and 4.
Regio-selective and stereo-selective sialylation of HO-3
of the galactose moiety in acceptor 20 with donor 10
was successfully accomplished because of the higher
nucleophilicity of HO-3 as compared to HO-2 and -4 of
this same moiety. The reaction entailed employment of
a defined configuration of the sialyl donor 10.
Trisaccharide acceptor 5 was then obtained by a one-
pot, two-step procedure in which compound 21 was
treated with Ac2O-pyridine in the presence of DMAP,
followed by treatment with 60% acetic acid. The (2!3)
linkage of 5 was confirmed by observation of a cross
peak between H0-3 and C00-2 in the HMBC spectrum.
The configuration of the glycoside of 5 was assigned
according to literature methods.11,12 and further con-
Since we needed to introduce a sulfate group at the
C-6 position of GlcNAc in the sialyl Le moiety in
hexasaccharide 3, the sialylated Lewis donor 9 which
carries the 2-naphthylmethyl (NAP) group at the C-6

J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3675
Scheme 1. Target oligosaccharide structures 1–3 and retrosynthetic analyses.
Scheme 2. The intermediate monosaccharide building blocks for target oligosaccharides 1–3.

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J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
firmed by observation of a strong cross peak between
H00-3a and C00-l in the HMBC spectrum of 5 because the
a-sialoside is expected to have a large heteronuclear
hydroxyl group in acceptor 5. Compound 1 was
obtained after global deprotection of 24 (Scheme 4).
8b,12
coupling constant, Jc-1, H-3a.
X-ray crystallography
Selective methylation of the 30-OH of disaccharide 22
was accomplished by a modified Hanessian procedure13
using methyl iodide and dibutyltin oxide. The di-
saccharide donor 4 was then obtained after complete
acetylation of 23 with acetic anhydride and pyridine.
The NIS-TfOH promoted glycosylation of trisaccharide
acceptor 5 with disaccharide donor 4 provided 24 as a
single glycosylation product in good yield (65%)
because of the much higher reactivity of the primary
Crystals of 10 were obtained by a slow evaporation of
the compound dissolved in anhydrous ethyl ether mix-
ture. The crystals are rectangular needle shaped. A
crystal of dimensions 0.25Â0.35Â0.l5 mm was used for
the present crystal structural investigation. The crystals
belong to the Orthorhombic system, space group
P212121. Table 1 gives the crystal data and structure
refinement parameters. Complete three-dimensional
data was collected on a CAD-4 computer-controlled
Scheme 3. (a) 1 M CH3ONa–CH3OH/CH2Cl2–CH3OH (1:1), À20 to À15 ꢀC, 20 min, 85%; (b) NIS-TfOH/CH3CN–CH2Cl2 (1:1), 3A-MS, À45 to
À40 ꢀC, 16 h, 45%; (c) Ac2O–pyridine (1:1), DMAP, rt, 12 h; (d) 60% HOAc, 65 ꢀC, 1.5 h, 72% in two steps.
Scheme 4. (a) (i) Bu3SnO/benzene, reflux, 4 h; (ii) ꢀCH3I-Bu4NI/benzene-DMF, 40–50ꢀꢀC, 4 days, 68%; (b) Ac2O–pyridine (1:1), DMꢀAP, rt, 12 h,
Ac2O–pyridine (1:1), DMAP, rt, 12 h; (f) 1 M CH3ONa–CH3OH/CH3OH–H2O, rt, 12 h, 37% in three steps.
.
90%; (c) NIS-TfOH/CH2Cl2, 4A-MS, À65 to À60 C, 65%; (d) LiI/pyridine, 120–125 C, 8 h, N2; (e) CH3OH–NH2NH2 H2O, 80–85 C, 6 h, then

J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3677
Scheme 5. (a) (i) Bu3SnO/benzene, reflux, 4 h; (ii) CH3I-Bu4NI/benzene-DMF, 40–50 ꢀC, 4 days, 65%; (b) Ac2O–pyridine (1:1), DMAP, rt, 12 h,
85%; (c) 60% HOAc, 65 ꢀC, 1 h; (d) NIS-TfOH/CH2Cl2, 4A-MS, À65 to À60 ꢀC, 49%; (e) DDQ/CH2Cl2–CH3OH (4:1), rt, 20 h, 70%; (f) LiI/
ꢀ
ꢀ
.
pyridine, 120–125 C, 8 h, N2; (g) CH3OH–NH2NH2 H2O, 80–85 C, 6 h, then Ac2O–pyridine (1:1), DMAP, rt, 12 h; (h) 1 M CH3ONa–CH3OH/
CH3OH–H2O, rt, 12 h, 33% in three steps.
diffractometer. 6192 reflections (2ymax=150ꢀ) were col-
lected by the o/2y method, out of which 5264 were
considered significant (1ꢁ2s). The crystal structure was
solved by a routine and straightforward application of
SHELX-96 program on a silicon graphics R10,000
computer. The structure was refined using SHELX-96
package14 of programs. All the hydrogen atoms were
located on a difference Fourier map. Refinements were
carried out with anisotropic thermal parameters for the
non-hydrogen atoms and isotropic thermal parameter
for the hydrogen atoms. The final reliability index
(R-factor) was 0.0712 for all the observed 5264 reflec-
tions and 0.0959 for all 6192 reflections with a goodness
of fit parameter S=1.072. Table 2 gives the final posi-
tional parameters for the non-hydrogen atoms in the
structure. Figure 2 gives an ORTEP10 diagram of the
molecule.
of pentasaccharide 27 to target 2 was accomplished in a
five-step sequence — (1) removal of the NAP group by
DDQ methodology, (2) removal of methyl from the
carboxyl group in sialic acid, (3) removal of Phth, (4)
Table 1. Crystal data and structure refinement for 10
Empirical formula
Color/shape
Formula weight
Temperature
Crystal system
Space group
Unit cell dimensions
C28H35O13NS
rectangular needle
625.6
22Æ3 ꢀC
Orthorhombic
P212121
a (A) 10.120 (3) a=90.0ꢀ
b (A) 16.684 (4) b=90.0ꢀ
c (A) 18.746 (4) g=90.0ꢀ
3165.1 (14) (A3)
4
Volume
Z
Density calculated
Absorption coefficient
Difftractometer
1.313 mg/m3
1.443 mmÀ1
CAD-4
The preparation of pentasaccharide 2 is outlined in
Scheme 5. Starting with known compound 25 (synthe-
sized from our protocol), regioselective methylation of
the 3-hydroxyl group of galactose in 25 with methyl
iodide in presence of dibutyltin oxide and tetrabutyl-
ammonium iodide in a mixture of benzene and DMF,
followed by complete acetylation in dry pyridine with
anhydrous acetic anhydride in the presence of a cataly-
tic amount of DMAP, provided the disaccharide com-
pound 26. Disaccharide acceptor 7 was obtained in
good yield (70%) by treatment of 26 with 60% HOAc at
65 ꢀC for 1.5 h. Complete regioselective glycosylation of
6-OH of acceptor 6 with trisaccharide donor 7 was
accomplished by an NIS–TfOH promoted system, pro-
viding pentasaccharide 27 as the only glycosylation
product in modes yield (49%). Complete deprotection
Radiation/wavelength
F(000)
Crystal size
Range for data collection
Reflections collected
Independent/observed reflections
Absorption correction
Range of relat. trasm. factors
Refinement method
Computing
CuKa/1.5418 A
1320
0.25Â0.35Â0.15 mm
0–150ꢀ
6192
5264 (Iꢁ2s)
Semi-empirical from psi scans
0.99 and 0.90
Full-matrix least squares on F2
SHELXS-96
Data/restraints/parameters
Goodness of fit on F 2
Function minimized
Final R indices
5264/0/517
1.072
ꢀ
ꢀ
ꢀ
ꢀ
Ã
2
Â
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Fo À ð1=kÞ Fc
0.0959 for all 6192 reflections
0.0712 for 5264 ½I ꢁ2ꢀIŠ
2.68Â10À7
Final extinction coefficient
Large diff. peaks and hole
Æ0.10 e/A3

3678
J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
complete acetylation with pyridine–Ac2O, and, (5)
complete deacetylation with 1 M sodium methoxide to
give 2 in 25% yield.
acetylation with Ac2O–pyridine (1:1) in the presence of
DMAP; (3) de-acetylation with 1 M sodium methoxide–
methanol solution.
Construction of hexasaccharide 3 is displayed in Scheme
6. The NIS-TfOH15 promoted glycosylation of the
primary hydroxyl group of disaccharide acceptor 8
with novel donor 9 gave 28 as a single glycosylation
product in good yield (65%) due to much higher reac-
tivity of the primary hydroxyl over the secondary axial
hydroxyl group in acceptor 8. Transformation of 28
into sulfated compound 29 was successfully achieved in
five successive steps — (1) acetylation of 28 with Ac2O–
pyridine, (2) careful removal of NAP protection with
our methodology,9 (3) sulfation, (4) removal of benzyl
under hydrogen atmosphere catalyzed by Pd/C (10%),
followed by (5) treatment with acetic anhydride in
pyridine. Target 3 was obtained from 29 by a global
deprotection procedure in three steps: (1) removal of
methyl with Lil–pyridine, (2) removal of phthalimido
The structure and purity of target oligosaccharides 1, 2
and 3 were fully characterized by a combination of 1D
1
(Fig. 3), and 2D-NMR techniques (2D H–1H DQF-
COSY, 2D ROESY) and ESIMS spectroscopy.
Our earlier studies show that core 2 branched structures
having 6-O-sulfated galactose in their Galb(1!3)Gal
NAc arm bind with selectins (L- and P-selectins). Our
preliminary binding studies with natural form hex-
asaccharide 3 indicate that this molecule can weakly
bind with L- and P-selectins. Examination of core 2
branched oligosaccharide analogues 1 and 2 as sub-
strates for Gal:6-O-sulfotransferase are underway.
Detailed biological studies of these oligosaccharides 1–3
will be reported elsewhere.
.
.
with CH3OH–NH2 NH2 H2O (5:1), then, complete
In summary, we have described a very effective protocol
for the construction of complex, core 2 branched, sialy-
lated and sulfated oligosaccharide structures 1 and 2
utilizing a strategy of regio- and stereoselective glycosy-
lation of unprotected or partially protected acceptors.
Additionally, a convergent method for the synthesis of
complex oligosaccharides through employment of a
versatile sialylated Lewis tetrasaccharide donor 9 has
been developed in our laboratory.
Table 2. Final fractional positional parameters for nonhydrogen
atoms with estimated standard deviations given in parentheses for 10
a
Atom
x
y
z
Ueq
S
O1
O2
O3
O4
O5
O6
O7
0.91206(10)
0.9129(2)
0.6807(4)
0.5850(4)
0.6593(2)
1.0262(4)
0.7845(5)
0.2682(3)
0.9846(2)
1.0738(4)
1.3652(6)
0.8401(3)
1.0765(6)
1.2103(3)
0.9135(3)
0.9709(3)
0.7145(4)
0.8277(3)
0.5742(4)
0.7729(4)
0.8497(5)
1.1102(4)
1.1627(3)
1.1084(5)
1.0623(3)
0.9968(4)
1.3682(4)
0.7652(3)
1.0767(5)
0.8619(3)
1.0135(3)
0.9026(5)
0.7986(8)
1.2415(8)
1.1109(6)
1.2892(10)
1.3088(8)
0.4712(5)
0.8560(8)
1.1725(7)
1.4747(11)
1.3369(8)
1.1806(6)
0.43862(6)
0.43154(12)
0.3206(2)
0.6219(2)
0.6198(2)
0.6506(2)
0.6670(2)
0.4550(2)
0.49965(14)
0.6128(2)
0.5325(4)
0.2882(2)
0.2036(3)
0.3006(2)
0.6532(2)
0.5090(2)
0.4822(2)
0.4234(2)
0.6427(2)
0.3375(2)
0.6995(2)
0.3573(2)
0.4380(2)
0.3302(3)
0.5060(2)
0.5588(2)
0.4992(3)
0.5681(2)
0.4083(3)
0.5713(2)
0.6806(2)
0.5460(3)
0.2057(3)
0.3112(5)
0.7406(3)
0.1696(4)
0.4432(9)
0.6966(4)
0.7891(3)
0.4670(4)
0.5000(7)
0.3663(8)
0.2235(3)
0.23055(4)
0.37678(11)
0.2832(3)
0.2384(2)
0.35010(14)
0.2985(2)
0.4999(2)
0.49579(14)
0.51308(11)
0.5567(2)
0.4162(3)
0.3590(2)
0.4911(6)
0.4542(2)
0.40243(13)
0.38502(15)
0.3212(2)
0.3186(2)
0.2993(2)
0.3184(2)
0.4547(2)
0.4692(2)
0.4467(2)
0.2637(2)
0.44932(15)
0.5624(2)
0.4725(3)
0.3299(2)
0.2456(2)
0.39177(15)
0.3563(2)
0.6218(2)
0.3593(3)
0.2721(3)
0.3833(4)
0.4426(5)
0.2460(5)
0.3290(4)
0.4500(4)
0.2370(3)
0.5256(10)
0.2642(5)
0.4651(3)
0.0534(3)
0.0400(5)
0.0866(12)
0.0741(9)
0.0492(6)
0.0639(8)
0.0756(10)
0.0511(6)
0.0396(5)
0.0810(11)
0.108(2)
O8
O9
O10
O11
O12
O13
N1
C1
C2
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C29
C30
0.0599(7)
0.133(3)
0.0603(7)
0.0413(6)
0.0361(6)
0.0480(7)
0.0419(6)
0.0529(8)
0.0521(8)
0.0551(9)
0.0503(8)
0.0407(6)
0.0635(10)
0.0367(6)
0.0500(8)
0.0605(10)
0.0423(6)
0.0557(9)
0.0378(6)
0.0463(7)
0.0684(11)
0.082(2)
0.097(2)
0.0778(14)
0.104(2)
0.121(4)
0.0788(15)
0.088(2)
0.076(2)
0.100(5)
0.113(3)
0.0743(13)
Figure 2. An ORTEP10 diagram showing the molecular structure of
compound 10. The thermal ellipsoids are drawn with 50% probability.
The coloring scheme followed is green (sulfur), red (oxygen), blue
(nitrogen), and white (carbon).
aUeq is defined as the trace of the orthogonalized Uij tensor.

J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3679
Scheme 6. (a) NIS-TfOH/CH2Cl2, 4A-MS, À65 to À60 ꢀC, 67%; (b) Ac2O–pyridine (1:1), DMAP, rt, 85%; (c) DDQ/CH2Cl2–CH3OH (4:1), rt, 22
h, 75%; (d) SO3–pyridine/pyridine, 0–5 ꢀC, 8 h, 85%; (e) Pd/C (10%), H2, CH2Cl2–CH3OH, rt, 6 h, then, Ac2O–pyridine (1:1), DMAP, rt, 12 h; (f)
ꢀ
ꢀ
.
LiI/pyridine, 120–125 C, 8 h, N2; (g) CH3OH–NH2NH2 H2O, 80–85 C, 6 h, then Ac2O–pyridine (1:1), DMAP, rt, 12 h; (h) 1 M CH3ONa–CH3OH/
CH3OH–H2O, rt, 12 h, 37% in three steps.
Figure 3. 600 MHz 1D 1H NMR of oligosaccharides: 1 (D2O), 2 (D2O+CD3OD) and 3 (D2O) recorded at 303.0 K.
Experimental
exposure to UV light or by spraying with 10% H2SO4,
and 0.2% p-anisaldehyde in a solution of ethanol and
heating or both. Solutions were concentrated under
reduced pressure at <40 ꢀC. The silica gel used for col-
umn chromatography was Baker Analyzed (60–200
General procedures
TLC was conducted on glass plates precoated with a
0.25-mm layer of silica gel 60 F-254 (Analtech GHLF
uniplates). The components were visualized either by
mesh). H NMR spectra were recorded at 30 ꢀC with
1

3680
J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
1
either a Bruker AMX 400 (400 MHz) or AMX 600
(600 MHz) spectrometer. The values of d (ppm) are
given relative to internal Me4Si (d=0) for solutions in
CDCl3, CD2Cl2, CD3OD. 13C NMR spectra were
recorded at 303 K with a Bruker AMX 400 (100.6 MHz)
spectrometer using CDCl3 (77.0 ppm), CD2Cl2 (54.15
ppm), CD3OD (49.15 ppm), acetone-d6 (206.0 or 29.8
ppm) as reference. First-order chemical shifts and cou-
pling constants (J/Hz) were obtained from 1D spectra
and assignments of protons resonance were based on
white solid. H NMR (CDCl3, 400 MHz): d=7.81–7.35
(m, 10H, ArH), 6.40 (d, 1H, J=8.4 Hz, NHAc), 5.60–
5.40 (m, 3H, benzylidene proton, H00-4, H00-8), 5.21 (d,
1H, J=3.6 Hz, H-1), 5.10 (dd, 1H, H00-7), 4.95 (dd, 1H,
H00-6), 4.80–4.65 (m, 2H, H-2, OCHAPh), 4.55 (d, 1H,
OCHBPh, Abq), 4.40–4.20 (m, 7H, H00-1, H-4, H00-9b,
H00-5, H0-6b, H-6b, H0-6a), 4.12 (dd, 1H, H0-3) 4.00 (dd,
1H, H-6a), 3.95–3.80 (m, 5H, H00-9a, H-3, COOCH3),
3.70–3.60 (m, 3H, H0-2, H0-5, H-5), 3.55 (t, 1H, H0-4),
2.85 (dd, 1H, J=4.6, 12.4 Hz, H00-3e), 2.38 (s, 3H,
NAc), 2.30 (s, 3H, NAc), 2.12 (s, 3H, Ac), 2.10 (s, 3H,
Ac), 2.00 (s, 3H, Ac), 1.98 (s, 3H, Ac), 1.89 (s, 3H, Ac),
1.20 (s, 9H, t-Bu); 13C NMR (CDCl3, 100.6 MHz):
d=178.10 (C¼O), 174.52 (C¼O), 171.20 (C¼O), 170.98
(C¼O), 170.65 (C¼O), 170.33 (C¼O), 169.93 (C¼O),
168.33 (C¼O), 138.01, 137.65, 129.04, 128.71, 128.57,
128.42, 128.29, 128.26, 126.79, 106.47, 101.26, 98.56,
97.38, 77.76, 77.54, 76.91, 76.20, 72.18, 70.63, 70.09,
69.47, 69.32, 68.95, 68.26, 67.94, 67.49, 66.76, 63.60,
63.03, 62.65, 56.69, 53.30, 48.77, 38.70, 27.39 (3CH3),
23.42 (NAc), 21.38 (Ac), 21.05 (Ac), 20.94 (Ac), 20.72
(Ac). Anal. calcd for C56H71O25N2: C, 57.38, H, 6.11,
N, 2.39. Found: C, 56.62, H, 5.93, N, 2.23.
1
2D DQF H–1H COSY and 2D ROESY. Two-dimen-
sional double-quantum filtered phase-sensitive 1H–1H
1
correlated spectra (DQF H–1H COSY) and rotating-
frame nuclear Overhauser enhancement spectroscopy
(ROESY) were recorded at 303 K using a Bruker AMX
400 (mixing time of 400 ms). All samples submitted for
elemental analyses were dried for 48 h under vacuum
over P2O5 at room temperature. Elemental analyses
were performed by Robertson Laboratory, Madison,
NJ, USA. Dichloromethane (CH2Cl2) and 1,2-dichlor-
oethane were kept over 4 A molecular sieves. Pyridine
was redistilled over potassium hydroxide.
Benzyl (6-O-trimethylacetyl-ꢀ-D-galactopyranosyl)-(1!3)-
4,6-O-benzylidene-2-acetamido-2-deoxy-ꢁ-D-galactopyr-
anoside (20).16 1H NMR (CDCl3, 400 MHz): d=7.54–
7.48 (m, 2H, ArH), 7.40–7.26 (m, 8H, ArH), 5.83 (d,
1H, J=9.2 Hz, NHAc), 5.12 (s, 1H, benzylidene pro-
ton), 5.08 (d, 1H, J 4.1 Hz, H-1), 4.71 (d, 1H, J=11.4
Hz, OCHAPh, ABq), 4.64 (dd, 1H, H-2), 4.55 (d, 1H,
J=12.2 Hz, OCHBPh, ABq), 4.22–4.10 (m, 4H), 3.97
(dd, 1H), 3.77 (dd, 1H, J=3.3, 10.9 Hz), 3.65 (d, 1H,
J=2.2 Hz, H0-4), 3.58–3.56 (m, 2H), 3.50 (t, 1H, J=6.2
Hz), 3.32 (dd, 1H), 1.91 (s, 3H, Ac), 1.20 (s, 9H, t-Bu);
13C NMR (CDCl3, 100.6 MHz): d=178.61 (C¼O),
174.35 (C¼O), 137.94, 137.12, 128.63, 128.63, 128.47,
126.96, 105.81, 101.43, 98.16, 76.17, 73.16, 72.87, 70.59,
69.24, 68.84, 63.55, 48.43, 38.89, 27.39 (3CH3), 23.68
Benzyl[methyl(N-acetyl-5-diacetamido-4,7,8,9-tetra- O-
acetyl-3,5-dideoxy-D-glycero-ꢁ-D-galacto-non-2-ulopyra-
nosyl)onate]-(2!3)-(6-O-trimethylacetyl-2,4-O-di-acetyl-
ꢀ-d-galactopyranosyl)-(1!3)-2-acetamido-2-deoxy-ꢁ-d-
galactopyranoside (5). A solution of compound 21 (300
mg, 0.26 mmol) and DMAP (3 mg) in acetic anhydride
(3 mL) and dry pyridine (3 mL) was stirred at room
temperature overnight. The reaction mixture was con-
centrated to a crude residue which was passed through a
short column of silica gel eluted with dichloromethane/
methanol (40:1) to give an amorphous solid. This pro-
duct was treated with 60% acetic acid at 60–65 ꢀC for
1.5 h and concentrated then passed through a short
column of silica gel eluted with dichloromethane/
methanol (25:1) to give a pure compound 5 (200 mg,
72%) as an amorphous solid. 1H NMR (CDCl3,
400 MHz): d=7.37–7.31 (m, 5H, ArH), 6.42 (d, 1H,
J=8.4 Hz, NHAc), 5.65 (ddd, 1H, H00-8), 5.54 (ddd, 1H,
H00-4), 5.11–5.07 (m, 2H, H00-7, H-1), 5.05–4.99 (m, 2H,
H0-2, H0-4), 4.71–4.68 (m, 2H, PhCHAO, H0-1), 4.63–
4.56 (m, 3H, H00-6, H0-3, H-2), 4.46 (d, 1H, Jgem=12.0
Hz, PhCHBO, ABq), 4.36–4.30 (m, 2H, H0-5, H00-9b),
4.17 (d, 1H, J=2.8 Hz, H-4), 4.11 (m, 1H, H-6b), 4.00–
3.90 (m, 2H, H-6a, H0-6b), 3.90–3.76 (m, 7H, H0-5, H-5,
COOCH3, H0-6a, H-3), 2.66 (dd, 1H, J=5.2, 12.6 Hz,
H00-3e), 2.35 (s, 3H, NAc), 2.25 (s, 3H, NAc), 2.20 (s,
3H, Ac), 2.17 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.06 (s, 3H,
Ac), 2.05 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.96 (s, 3H, Ac),
1.92 (s, 3H, Ac), 1.82 (s, 3H, Ac), 1.58 (t, 1H,
Jgem=12.4 Hz, H00-3a), 1.19 (s, 9H, t-Bu); 13C NMR
(CDCl3, 100.6 MHz): d=177.76 (C¼O), 174.11 (C¼O),
173.74 (C¼O), 171.55 (C¼O), 171.24 (C¼O), 170.05
(C¼O), 169.99 (C¼O), 169.86 (C¼O), 169.80 (C¼O),
167.97 (C¼O), 137.39, 128.75, 128.64, 128.26, 102.76,
97.57, 96.66, 78.59, 71.41, 70.74, 70.22, 69.93, 69.90,
69.36, 69.28, 69.23, 67.57, 67.41, 67.16, 67.01, 63.23,
63.01, 61.06, 55.88, 53.10, 48.35, 38.37, 28.17 (NAc),
27.14 (3 CH3), 26.82 (NAc), 23.15 (NAc), 21.52 (Ac),
21.12 (Ac), 21.02 (Ac), 20.79 (Ac). Anal. calcd for
.
(NAc). Anal. calcd for C33H43O12N 3H2O: C, 56.64, H,
7.06, N, 2.00. Found: C, 56.11, H, 6.41, N, 1.68.
Benzyl[methyl(N-acetyl-5-diacetamido-4,7,8,9-tetra- O-
acetyl-3,5-dideoxy-D-glycero-ꢁ-D-galacto-non-2-ulopyr-
anosyl)onate]-(2!3)-(6-O-trimethylacetyl-ꢀ-D-galacto-
pyranosyl)-(1!3)-2-acetamido-4,6-O-benzylidene-2-deoxy-
ꢁ-D-galactopyranoside (21). A solution of compound 10
(582 mg, 0.93 mmol), compound 20 (540 mg, 0.84
mmol), N-iodosuccinimide (NIS, 625 mg, 2.78 mmol) in
dry dichloromethane–acetonitrile (20 mL, 1:1) contain-
ing 3 A MS (2 g) was stirred at À70 to À50 ꢀC for 2 h
under N2 atmosphere. Trifluoromethanesulfonic acid
(TfOH) (100 mL) in 2 mL dry acetonitrile was then
added dropwise and stirring continued for 6 h. Addi-
tional portions of donor 10 (220 mg) and tri-
fluoromethanesulfonic acid (30 mL) were then added
and stirring was continued for a total of 16 h. The mix-
ture was neutralized with satd aq sodium bicarbonate.
Solids were filtered off and the organic layer washed
with satd aq NaHCO3, 10% Na2S2O3, water, dried
(Na2SO4) and concentrated under reduced pressure to a
crude product. The product was applied to a column of
silica gel eluted with dichloromethane/methanol (30:1)
to give a pure compound 21 (430 mg, 45%) as a glass

J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3681
C53H71O27N2: C, 54.49, H, 6.13, N, 2.40. Found: C,
54.98, H, 6.23, N, 2.42.
chloromethane containing 4 A MS (500–800 mg/mL)
was stirred at À65 to À60 ꢀC for 2 h under N2 atmos-
phere. Trifluoromethanesulfonic acid (TfOH) (25–32
mL/mmol NIS) in dry dichloromethane (2 mL) was then
added dropwise and stirring was continued at the same
temperature for 1.5–2 h. Reaction monitored by TLC.
The mixture was neutralized with satd aq sodium
bicarbonate, solids were filtered off and the organic
layer washed with satd aq NaHCO3, 10% Na2S2O3,
water, dried (Na2SO4) and concentrated under reduced
pressure. The resultant mixture was purified on a col-
umn of silica gel eluted with dichloromethane/methanol
to give pure product.
Phenyl(3-O-methyl-ꢀ-D-galactopyranosyl)-(1!4)-6-O-tri-
methylacetyl-2-deoxy-2-phthalimido-1-thio-ꢀ-D-glucopyr-
anoside (23). A solution of compound 22 (210 mg, 0.29
mmol) in dry methanol (10 mL) received dibutyltin
oxide (72 mg, 0.29 mmol) and was refluxed for 4 h. This
mixture was concentrated and the residue treated with
tetrabutylammonium iodide (62 mg, 0.44 mmol) and
methyl iodide (12.12 g, 54.86 mmol) in dry benzene/
DMF (1:1) (10 mL) at 40–45 ꢀC for 4 days. The mixture
was concentrated under reduced pressure and the crude
residue was applied to a column of silica gel and eluted
first with hexane/ethyl acetate (4:1), and then with hex-
ane/ethyl acetate (1:1) to give compound 23 (148 mg,
68%) as an amorphous solid. 1H NMR (CD3OD,
400 MHz): d=8.00–7.80 (m, 4H, ArH), 7.40–7.20 (m,
5H, ArH), 5.51 (d, 1H, J=8.4 Hz, H-1), 4.65 (dd, 1H),
4.40–4.30 (m, 3H), 4.10 (t, 1H), 4.00 (s, 1H), 3.82 (m,
1H), 3.78–3.54 (m, 4H), 3.51 (m, 3H), 3.44 (s, 3H,
OCH3), 3.10 (dd, 1H), 1.10 (s, 9H, t-Bu); 13C NMR
(CD3OD, 100.6 MHz): d=147.57, 147.36, 147.15,
135.85, 133.86, 133.49, 130.12, 129.22, 124.66, 124.39,
105.16 (C0-1), 84.97 (C-1), 84.34, 80.80, 78.69, 77.39,
72.40, 71.61, 66.23, 64.30, 64.28, 62.85, 57.56, 56.81,
Pentasaccharide (24). As an amorphous solid, yield
1
65%. H NMR (CDCl3, 600 MHz): d=7.80–7.10 (m,
9H, ArH), 5.70 (dd, 1H, J=8.3, 7.9 Hz, H000-3), 5.58–
5.49 (m, 2H, H00-4, H00-8), 5.47 (d, 1H, J=2.8 Hz, H0000-
4), 5.43 (d, 1H, J=8.7 Hz, H000-l), 5.14 (dd, 1H, J=2.6,
9.6 Hz, H00-7), 5.02 (d, 1H, J=3.2 Hz, H0-4), 4.92–4.85
(m, 2H, H0000-2, H0-2), 4.68 (d, 1H, J=7.3 Hz, H0-1),
4.64–4.56 (m, 3H, H00-6, H0000-1, H0-3), 4.49 (d, 1H,
J=3.0 Hz, H-1), 4.38–4.32 (m, 2H, H-2, H00-5), 4.28–
4.20 (m, 2H, H00-9b, H0000-2), 4.20–3.84 (m, 15H, H00-9b,
PhCHAO, H000-2, H0-6b, H0-6a, H-6b, H0000-5, H-4, H000-4,
H0-5, H-5, H00-9a, COOCH3), 3.76 (d, 1H, Jgem=11.0
Hz, PhCHBO, ABq), 3.73–3.68 (m, 2H, H-6a, H-3), 3.52
(dd, 1H, H0000-3), 3.32 (s, 3H, OCH3), 2.59 (dd, 1H,
J=5.1, 12.5 Hz, H00-3e), 2.35 (s, 3H, NAc), 2.34 (s, 3H,
NAc), 2.33 (s, 3H, NAc), 2.20 (s, 3H, Ac), 2.18 (s, 3H,
Ac), 2.12 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.06 (s, 3H, Ac),
2.04 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.90
(s, 3H, Ac), 1.83 (s, 3H, Ac), 1.48 (t, 1H, J=11.7 Hz,
H00-3a), 1.28 (s, 9H, t-Bu), 1.16 (s, 9H, t-Bu). Anal. calcd
for C83H108O41N3: C, 55.27, H, 6.04, N, 2.33. Found: C,
55.15, H, 6.13, N, 2.15.
.
27.86 (3CH3). Anal. calcd for C32H39O12NS H2O: C,
56.54, H, 6.08, N, 2.07, S, 4.72. Found: C, 56.77, H,
5.49, N, 1.99, S, 4.83.
Phenyl(2,4,6-tri-O-acetyl-3-O-methyl-ꢀ-D-galactopyrano-
syl)-(1!4)-4-O-acetyl-6-O-trimethyl-acetyl-2-deoxy-2-
phthalimido-1-thio-ꢀ-D-glucopyranoside (4). A solution
of compound 23 (140 mg, 0.19 mmol) and DMAP (5
mg) in acetic anhydride (6 mL) and dry pyridine (6 mL)
was stirred at room temperature overnight. The reaction
mixture was concentrated to a crude mixture, which was
applied to a short column of silica gel eluted with hex-
ane/ethyl acetate (1:1) to give a pure compound 4 (90%)
A general procedure B for deprotection of compound 24,
27, and 29
1
as an amorphous solid. H NMR (CDCl3, 400 MHz):
d=7.78–7.72 (m, 2H, ArH), 7.41–7.36 (m, 2H, ArH),
7.28–7.20 (m, 3H, ArH), 5.80–5.70 (m, 2H, H-3, H-1),
5.42 (d, 1H, J=2.8 Hz, H0-4), 4.98 (dd, 1H, H0-2), 4.52
(dd, 1H), 4.42 (d, 1H, J=7.8 Hz, H0-1), 4.30–4.15 (m,
2H), 4.10–4.00 (m, 2H), 3.90–3.70 (m, 3H), 3.35 (s, 3H,
OCH3), 3.25 (dd, 1H), 2.28 (s, 3H, Ac), 2.20 (s, 3H, Ac),
2.15 (s, 3H, Ac), 1.84 (s, 3H, Ac), 1.10 (s, 9H, t-Bu); 13C
NMR (CDCl3, 100.6 MHz): d=178.09 (C¼O), 174.28
(C¼O), 170.61 (C¼O), 170.36 (C¼O), 169.97 (CO),
169.36 (C¼O), 167.44 (C¼O), 134.63, 134.38, 133.26,
131.91, 131.48, 131.44, 129.10, 128.44, 123.97, 123.75,
101.29 C0-1), 83.22 (C-1), 80.10, 76.91, 76.74, 71.99,
71.20, 70.80, 65.19, 62.76, 61.69, 58.27, 54.09, 39.05
[C(CH3)3], 27.45 (3CH3), 20.91 (Ac), 20.85 (Ac), 20.68
To a solution of compound 24 (32 mg, 17 mmol) in dry
pyridine (3 mL), was added lithium iodide (Lil, 50 mg,
0.37 mmol). The mixture was refluxed at 120–125 ꢀC for
6 h under N2 atmosphere. The dark yellow solution was
then concentrated to dryness and co-evaporated with
toluene to a corresponding carboxylic acid as a dark
yellow amorphous solid which was used directly for the
next reaction. A solution of the above in methanol (5
.
mL), was treated with NH2–NH2 H2O (1 mL) for 4 h at
80–85 ꢀC, the mixture was concentrated under reduced
pressure, co-evaporated with toluene then acetylated
with acetic anhydride–pyridine (1:1) in the presence of a
catalytic amount of DMAP at room temperature over-
night. The acetylated mixture was concentrated and
passed through a short column of silica gel eluted with
dichloromethane/methanol (10:1) to give a bright yellow
film. To a solution of this bright yellow film in metha-
nol–water (1 mL, 1:1), was added a catalytic amount of
1 M sodium methoxide (150 mL). The mixture was stir-
red at room temperature for 48 h and concentrated under
reduced pressure to give a crude mixture which was then
applied to a short column of silica gel eluted with
n-C3H7OH/HOAc/H2O (3:1:1) to give a pure compound.
.
(Ac). Anal. calcd for C40H47O16NS H2O: C, 56.66, H,
5.88, N, 1.65, S, 3.78. Found: C, 55.97, H, 5.03, N, 1.45,
S, 3.69.
Glycosylation procedure for preparation of 24, 27, and
28 with NIS–TfOH promoter
A solution of acceptor (1 mmol), donor (1.05–1.1
mmol), N-iodosuccinimide (NIS, 3.0 mmol) in dry di-

3682
J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
Deprotected pentasaccharide (1). See general procedure
B, (11 mg, 57%) as an amorphous solid. 1H NMR
(D2O, 600 MHz) d=7.40–7.20 (m, 5H, ArH), 4.88 (d,
1H, J=3.6 Hz, H-1), 4.61 (d, 1H, J=11.4 Hz,
PhCHAO, ABq), 4.45 (d, 1H, J=8.4 Hz, H000-l), 4.41–
4.36 (m, 3H, H0-1, PhCHBO, H0000-1), 4.18 (dd, 1H, H-2),
4.14–4.10 (dd, 2H, H-4, H0000-4), 4.06 (dd, 1H, H-5),
3.96–3.92 (m, 3H, H0-3, H-6b, H-3), 3.87 (dd, 1H, H000-
2), 3.81 (d, 1H, J=2.3 Hz, H0-4), 3.75–3.40 (m, 20H, H-
6a, H00-4, H0-5, H0000-2, H0-2), 3.34 (s, 3H, OCH3), 3.26
(dd, 1H, J=3.1, 10.0 Hz, H0000-3), 2.64 (dd, 1H, J=4.6,
2.4 Hz, H00-3e), 1.92 (s, 3H, Ac), 1.85 (s, 3H, Ac), 1.81
(s, 3H, Ac), 1.67 (t, 1H, J=11.8 Hz, H00-3a); 13C NMR
(D2O, 100.6 MHz) d=182.40 (C¼O), 176.21 (C¼O),
175.60 (C¼O), 175.40 (C¼O), 138.03, 129.84, 129.69,
129.47, 105.45, 103.87, 102.58, 100.81 (C00-2), 97.32,
82.78, 79.50, 78.23, 76.75, 76.35, 75.86, 73.91, 73.59,
72.88, 71.65, 70.87, 70.68, 70.50, 70.19, 69.78, 69.43,
69.20, 68.52, 65.22, 63.62, 62.18, 62.08, 61.16, 57.34,
56.20, 52.81, 40.85 (C00-3), 23.45 (NAc), 23.28 (NAc),
23.12 (NAc); ESIMS (m/z) (negative ion mode)
C47H73O29N3: 1143.3; found: 1142 [MÀH]À.
5H, ArH), 7.41–7.32 (m, 8H, ArH), 5.70 (dd, 1H,
J=8.3, 7.9 Hz, H000-3), 5.59–5.48 (m, 2H, H00-4, H00-8),
5.48 (d, 1H, J=2.9 Hz, H0000-4), 5.45 (d, 1H, J=8.7 Hz,
H000-l), 5.15 (dd, 1H, J=2.8, 9.6 Hz, H00-7), 5.03 (d, 1H,
J=2.8 Hz, H0-4), 4.93–4.86 (m, 2H, H0000-2, H0-2), 4.69
(d, 1H, J=7.3 Hz, H0-1), 4.65–4.57 (m, 3H, H00-6, H0000-l,
H0-3), 4.48 (d, 1H, J=3.1 Hz, H-1), 4.39–4.20 (m, 7H,
H-2, H00-5, H00-9b, H0000-2), 4.21–3.85 (m, 15H, H00-9b,
PhCHAO, H000-2, H0-6b, H0-6a, H-6b, H0000-5, H-4, H000-4,
H0-5, H-5, H00-9a, COOCH3), 3.75 (d, 1H, Jgem=12.6
Hz, PhCHBO, ABq), 3.74–3.69 (m, 2H, H-6a, H-3), 3.53
(dd, 1H, H0000-3), 3.34 (s, 3H, OCH3), 2.61 (dd, 1H,
J=4.8, 12.6 Hz, H00-3e), 2.36 (s, 3H, NAc), 2.34 (s, 3H,
NAc), 2.32 (s, 3H, NAc), 2.21 (s, 3H, Ac), 2.16 (s, 3H,
Ac), 2.13 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.06 (s, 3H, Ac),
2.05 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.91
(s, 3H, Ac), 1.85 (s, 3H, Ac), 1.51 (t, 1H, J=11.7 Hz,
H00-3a), 1.27 (s, 9H, t-Bu). Anal. calcd for
C92H113O42N3: C, 57.16, H, 5.89. Found: C, 57.70, H,
5.95.
Deprotection of pentasaccharide 27 to target
oligosaccharide (2)
Benzyl(2,4-O-di-acetyl-3-O-methyl-ꢀ-D-galactopyranosyl)-
(1!3)-2-acetamido-2-deoxy-ꢁ-D-galactopyranoside (7).
To a solution of compound 251b (370 mg, 0.57 mmol)
in dry methanol (15 mL) was added dibutyltin oxide
(142 mg, 0.57 mmol), and refluxed for 2 h. The reaction
mixture was concentrated to a crude residue which was
treated with tetrabutylammonium iodide (142 mg) and
methyl iodide (54 uL, 0.86 mmol) in dry benzene–DMF
(1:1) (10 mL) at 40–43 ꢀC for 4–5 days. The reaction
mixture was concentrated to a crude residue which was
passed through a short of column of silica gel and eluted
with dichloromethane/methanol (10:1) to give a pure
compound (240 mg) in 56% yield as an amorphous
solid. A solution of the above compound 26 (240 mg)
was treated with a mixture of dry pyridine (4 mL) and
anhydrous acetic anhydride (4 mL) in the presence of
DMAP (5 mg) at room temperature overnight. The
reaction was concentrated under reduced pressure to a
crude residue which was purified by a short column of
silica gel eluted with dichloromethane/methanol (40:1)
to give a pure acetylated compound 26 (250 mg) which
was treated with 60% HOAc at 65 ꢀC for 1 h. The
reaction mixture was concentrated under reduced pres-
sure to a crude mixture which was passed through a
short column of silica gel eluted with dichloromethane/
methanol (20:1) to give a pure compound 7 (147 mg) in
To a solution of compound 27 (63 mg, 0.037 mmol) in a
mixture of dichloromethane–methanol (5 mL, 4:1) was
added DDQ (12 mg). The reaction mixture was stirred
at room temperature for 20 h. The reaction was con-
centrated under reduced pressure to a crude residue
which was then redissolved in dichloromethane (50 mL)
and washed with satd NaHCO3 solution (3Â50 mL).
The organic phase was dried with Na2SO4 and con-
centrated to a crude residue which was purified by a
short column of silica gel eluted with dichloromethane–
methanol (20:1) to give a pure compound (41 mg) in
70% yield as amorphous solid. The compound thus
obtained was subjected to general procedure B to give
target oligosaccharide 2 (8.0 mg) as an amorphous solid.
1H NMR (D2O, 600 MHz) d=7.48–7.40 (m, 5H, ArH),
4.96 (d, 1H, J=3.3 Hz, H-1), 4.70 (d, 1H, J=11.3 Hz,
PhCHAO, ABq), 4.54 (d, 1H, J=8.6 Hz, H00-1), 4.52 (d,
1H, J=7.9 Hz, H000-l), 4.50 (d, 1H, J=11.3 Hz,
OCHBPh, Abq), 4.44 (d, 1H, J=7.8 Hz, H0-1), 4.30 (dd,
1H, J=3.4, 11.2 Hz, H-2), 4.20 (d, 1H, J=2.8 Hz, H-4),
4.17 (d, 1H, J=3.1 Hz, H0-4), 4.13 (dd, 1H), 4.10 (dd,
1H, J=3.0, 9.9 Hz, H000-3), 4.06–3.96 (m, 3H, H-6a, H-
3,), 3.95 (d, 1H, J=2.6 Hz, H000-4), 3.92–3.48 (m, 22H,
H-6b, H0000-4, H000-2, H0-2), 3.40 (s, 3H, OCH3), 3.29 (dd,
1H, H0-3), 2.75 (dd, 1H, J=4.6, 12.4 Hz, H000-3e), 2.04
(s, 3H, Ac), 1.98 (s, 6H, 2Ac), 1.79 (t, 1H, J=11.9 Hz,
H00-3a); 13C NMR (D2O, 100.6 MHz) d=182.50 (C¼O),
176.50 (C¼O), 175.50 (C¼O), 175.10 (C¼O), 138.20,
130.00, 129.98, 129.80, 105.60, 103.80, 102.65, 101.00
(C00-2), 97.45, 82.80, 79.62, 78.20, 76.65, 76.40, 76.10,
75.98, 74.10, 73.62, 70.89, 70.80, 70.78, 70.60, 70.58,
70.10, 69.49, 69.25, 68.80, 68.72, 65.21, 63.80, 62.20,
61.25, 56.15, 56.05, 53.00, 49.55, 40.98, 23.44 (NAc),
23.22 (NAc), 23.15 (NAc); ESIMS (m/z) (negative ion
mode) C47H73O29N3: 1143.3; found: 1141.9 [MÀH]À.
1
66% yield as an amorphous solid. H NMR (CDCl3,
400 MHz) d=7.41–7.29 (m, 5H, ArH), 5.48 (d, 1H,
J=3.1 Hz), 4.96 (dd, 1H, J=8.1, 8.0 Hz), 4.83 (d, 1H,
J=4.6 Hz), 4.76 (dd, 2H, PhCH2O), 4.71 (d, 1H, J=7.9
Hz), 4.50–4.45 (m, 2H), 4.15–4.09 (m, 3H), 3.95–3.87
(m, 3H), 3.76–3.70 (m, 2H), 3.51 (dd, 1H), 3.32 (s, 3H,
OCH3), 2.09 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.01 (s, 3H,
Ac), 1.95 (s, 3H, Ac). Anal. calcd for C28H39O14N: C,
54.81, H, 6.40. Found: C, 54.77, H, 5.90.
Pentasaccharide (27). The compound 27 (95 mg) was
obtained from acceptor 7 (67 mg) according to general
glycosylation procedure A in 49% yield as amorphous
Sialylated Lewis tetrasaccharide (9).9 TLC: Rf=0.48
(hexane/ethyl acetate 1:1), 1H NMR (DMF-d7,
600 MHz) d=7.84–7.60 (m, 8H, ArH), 7.52–7.36 (m,
1
solid. H NMR (CDCl3, 600 MHz): d=7.52–7.49 (m,

J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3683
6H, ArH), 7.12–7.00 (m, 17H, ArH), 5.57–5.50 (m, 3H,
H00-8, H00-4, H-1, J1,2=10.9 Hz), 5.19 (dd, 1H, J=2.3,
9.5 Hz, H00-7), 5.00 (d, 1H, J=3.8 Hz, H0-4), 4.97 (d,
1H, J1,2=9.1 Hz, H0-1), 4.91 (dd, 1H, H0-2), 4.87–4.85
(m, 2H, OCHAr, H000-1), 4.82–4.65 (m, 5H, OCHAr, H-
3, OCHAr, H0-3, H000-5), 4.62 (dd, 2H, OCH2Ar, ABq),
4.59–4.47 (m, 3H, H00-9b, OCHAr, H-2), 4.41 (d, 1H,
Jgem=12.1 Hz, OCHAr, ABq), 4.33–4.17 (m, 4H, H00-
9a, OCHAr, H00-6, H-4), 4.09 (dd, 1H, H00-5), 4.04–3.95
(m, 4H, H-6b, H0-6b, H0-5, H-6a), 3.92–3.78 (m, 6H,
H000-3, H0-6a, COOCH3, H000-2), 3.80 (dd, 1H, H000-5),
3.70 (dd, 1H, H-5), 3.62 (d, 1H, J=2.8 Hz, H000-4), 2.60
(dd, 1H, J=4.9, 12.3 Hz, H00-3e), 2.35 (s, 3H, Ac), 2.28
(s, 3H, Ac), 2.22 (s, 3H, Ac), 2.10 (s, 3H, Ac), 1.98 (s,
3H, Ac), 1.97 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.79 (s, 3H,
Ac), 1.58 (t, 1H, J=11.0 Hz, H00-3a), 1.25 (d, 3H, J=6.7
Hz, CH0003), 1.10 (s, 9H, t-Bu); 13C NMR (CDCl3,
100.6 MHz) d=177.31 (C¼O), 174.24 (C¼O), 173.81
(C¼O), 170.74 (C¼O), 170.38 (C¼O), 170.13 (C¼O),
169.86 (C¼O), 169.27 (C¼O), 167.99 (C¼O), 139.17,
138.88, 138.56, 136.17, 134.31, 133.47, 133.03, 132.51,
128.94, 128.31, 128.25, 128.23, 128.10, 128.04, 127.83,
127.81, 127.51, 127.29, 127.18, 127.03, 126.12, 125.94,
125.81, 125.67, 123.82, 99.79, 97.54, 96.79 (Cc-2), 84.07
(Ca-1), 80.02, 79.89, 77.69, 74.88, 74.79, 74.41, 73.60,
73.05, 73.03, 72.50, 71.90, 70.74, 70.35, 69.49, 68.47,
67.37, 67.30 (2C), 67.00, 66.65, 62.14, 60.12, 56.05,
55.60, 53.05, 38.65 [C(CH3)3], 38.55 [(CH2)00], 28.29
(Ac), 27.13 (CH3), 26.94 (Ac), 21.52 (Ac), 21.22 (Ac),
21.07 (Ac), 20.88 (Ac), 20.75 (Ac), 16.92 (CH0003). Anal.
calcd for C95H106O31N2S: C, 63.25, H, 5.92, N, 1.55, S,
1.78. Found: C, 63.17, H, 6.40, N, 1.40, S, 1.70.
99.71, 99.23, 98.25, 97.47, 96.72, 79.91, 78.48, 75.39,
75.27, 74.59, 74.39, 73.91, 72.83, 72.41, 72.33, 71.51,
71.44, 71.18, 70.85, 70.68, 70.42, 69.45, 69.28, 69.25,
69.11, 68.72, 68.18, 67.49, 67.45, 67.43, 67.08, 68.83,
66.52, 63.13, 60.89, 60.19, 56.65, 55.92, 54.39, 53.08,
48.25, 38.37, 27.21 (3CH3), 23.24 (Ac), 21.55 (Ac), 21.15
(Ac), 21.05 (Ac), 20.81 (Ac), 20.69 (Ac), 16.81 (CH3).
Anal. calcd (%) for C115H141O46N3: C, 59.94, H, 6.17,
N, 1.82. Found: C, 59.64, H, 5.86, N, 1.52.
Deprotected hexasaccharide (3). A solution of com-
pound 28 (292 mg, 0.13 mmol) and DMAP (4 mg) in
acetic anhydride (3 mL) and dry pyridine (3 mL) was
stirred at room temperature overnight. The reaction
mixture was concentrated to a crude residue which was
passed through a short column of silica gel eluted with
dichloromethane/methanol (40:1) to give a pure com-
pound (264 mg, 87%). To a solution of this compound
(264 mg, 0.112 mmol) in a mixture of dichloromethane–
methanol (8 mL, 4:1) was added DDQ (38 mg) and
stirred at room temperature overnight. Another addi-
tional portion of DDQ (20 mg) was added and stirred
for a total 22 h. The reaction was concentrated to a
crude residue which was dissolved in dichloromethane
and washed with satd NaHCO3, water, and dried over
Na2SO4. The organic layer was concentrated to a crude
mixture (95 mg) which was treated with SO3–pyridine
complex (15 mg) in dry pyridine (2 mL) at 0 ꢀC over-
night. The reaction mixture was concentrated to a crude
residue which was purified by a short column of silica
gel eluted with dichloromethane/methanol (20:1) to give
a pure compound (64 mg). A solution of the above
obtained compound (82 mg) and Pd/C(10%) (80 mg) in
a mixture of dichloromethane–methanol (10 mL) was
stirred overnight under H2 atmosphere. The filtrate was
concentrated to a crude residue which was treated with
acetic anhydride (3 mL) and pyridine (3 mL) in the
presence of DMAP (3 mg) at room temperature over-
night. The reaction mixture was concentrated to a crude
product, which was passed through a short column of
silica gel eluted with CH2Cl2/MeOH (20:1) to give pure
compound 29 (60 mg) as an amorphous solid which was
directly subjected to general deprotection procedure B
to give compound 3 (14 mg). 1H NMR (D2O,
600 MHz): d=5.13 (d, 1H, J=3.1 Hz, H00000-1), 4.81 (dd,
1H, H00000-5), 4.78 (d, 1H, J=3.6 Hz, H-1), 4.66–4.60
(dd, 2H, H000-1, H00-1), 4.48 (d, 1H, J=7.5 Hz, H0-1),
4.40 (t, 2H, H00-6b, H00-6a), 4.33 (dd, 1H, H-20, 4.22 (d,
1H, H-4), 4.12 (dd, 2H, H-5), 4.08–3.06 (m, 26H, H-6b,
H00-4, H-3, H00-2, H00-3, H-6a), 3.56–3.50 (m, 2H, H0-2,
H000-2), 3.38 (s, 3H, OCH3), 2.78 (dd, 1H, H0000-3e), 2.06
(s, 3H, Ac), 2.02 (s, 3H, Ac), 1.82 (t, 1H, H0000-3a), 1.20
(d, 3H, J=5.9 Hz, CH000003); 13C NMR (D2O,
100.6 MHz): d=180.58 (C¼O), 174.68 (C¼O), 174.25
(C¼O), 173.89 (C¼O), 104.36, 101.35, 100.90, 99.36,
(C0000-2), 98.25, 97.85, 76.92, 75.15, 74.61, 74.50, 74.44,
72.62, 72.41, 72.55, 72.41, 72.25, 71.62, 71.06, 70.41,
70.35, 69.12, 69.05, 68.91, 68.64, 68.29, 68.10, 68.05,
67.82, 67.45, 66.97, 66.40, 65.65, 62.27, 61.16, 60.63,
55.34, 54.68, 51.48, 48.24, 39.58, 21.96 (NAc), 21.76
(NAc), 21.72 (NAc), 14.98 (CH000003); ESIMS (m/z)
(negative ion mode) calcd for C46H76O36N3SNa [M]:
1301; found 1278.3 [MÀNa]À, 638.9 [MÀ2H]À.
Hexaccharide (28). As an amorphous solid yield 67%.
1H NMR (CDCl3, 600 MHz): d=7.95–7.88 (m, 4H,
ArH), 7.71–7.68 (m, 2H, ArH), 7.55–7.51 (m, 4H, ArH),
7.29–7.10 (m, 16H, ArH), 6.09 (d, J=9.6 Hz, 1H,
NHAc), 5.66 (ddd, 1H, H0000-8), 5.58 (ddd, 1H, H0000-4),
5.15–5.11 (m, 3H; H000-1, H0000-7, H0000-4), 5.06–4.99 (m,
3H; H0-2, H0000-2, OCHAr, ABq), 4.86–4.74 (m, 4H; H-4,
J3,4=3.6 Hz, OCHAr, ABq, H00-3, H00-1, J1,2=8.0 Hz),
4.73–4.71 (m, 2H, J1,2=5.2 Hz; H-1, H0-1), 4.68–4.57
(m, 3H; OCHAr, Jgem=12.0 Hz, H0000-6, OCHAr, Jgem
11.6 Hz, ABq), 4.46 (d, Jgem=12.8 Hz, 1H; OCHAr,
ABq ), 4.38–4.18 (m, 7H; H00000-1, H00000-4, H000000-5, H0000-9b,
H-2, H000-4), 4.12–3.90 (m, 14H; H00-6b, H -6b, H-6b,
H
00000-3, H000-6b, H1-6a, H00-6a, COOCH3, H000-6a, H0000-
9a, H0-5, H-3), 3.73–3.56 (m, 2H; H-6a, H00000-2), 3.36 (t,
J=6.8, 7.2 Hz, 1H; H00-5), 2.84 (s, 3H; OCH3), 2.67 (dd,
J=5.2, 12.8 Hz, 1H; H0000-3e), 2.38 (s, 3H; Ac), 2.35 (s,
3H; Ac), 2.26 (s, 3H; Ac), 2.19 (s, 3H; Ac), 2.10 (s, 3H;
Ac), 2.07 (s, 3H; Ac), 2.04 (s, 3H; Ac), 1.97 (s, 3H; Ac),
1.95 (s, 3H; Ac), 1.94 (s, 3H; Ac), 1.93 (s, 3H; Ac), 1.91
(s, 3H; Ac), 1.59 (t, J=12.4 Hz, 1H; H0000-3a), 1.21 (s,
12H; t-Bu, CH300000), 1.20 (s, 9H, t-Bu); 13C NMR
(CDCl3, 100.6 MHz): d=178.70 (C¼O), 174.20 (C¼O),
173.70 (C¼O), 173.17 (C¼O), 171.38 (C¼O), 171.16
(C¼O), 170.38 (C¼O), 170.17 (C¼O), 170.08 (C¼O),
170.03 (C¼O), 169.88 (C¼O), 169.78 (C¼0), 169.70
(C¼O), 169.61 (C¼O), 168.82 (C¼O), 167.96 (C¼O),
139.11, 138.90, 138.51, 135.41, 134.15, 133.21, 128.70,
128.31, 128.27, 128.20, 128.09, 127.95, 127.51, 127.28,
127.21, 127.19, 126.83, 126.45, 126.13, 125.99, 102.62,

3684
J. Xia et al. / Bioorg. Med. Chem. 10 (2002) 3673–3684
3. (a) Varki, A. Glycobiology 1993, 3, 97. (b) Dwek, R. D.
Acknowledgements
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This work was supported by Grants Nos. CA35329,
CA63218 and P30CA16056, all awarded by National
Cancer Institute, and in part by the Charlotte Geyer
Foundation.
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