Chemical preparation of sialyl Lewis x using an enzymatically synthesized sialoside building block

Article abstract of DOI:10.1016/j.carres.2008.06.020

The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycosylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide. Combining the advantages of highly efficient enzyma

Full text of DOI:10.1016/j.carres.2008.06.020

Carbohydrate Research 343 (2008) 2863–2869
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemical preparation of sialyl Lewis x using an enzymatically synthesized
sialoside building block
*
Hongzhi Cao, Shengshu Huang, Jiansong Cheng, Yanhong Li, Saddam Muthana, Bryan Son, Xi Chen
Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 May 2008
Received in revised form 20 June 2008
Accepted 26 June 2008
Available online 4 July 2008
The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycos-
ylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide.
Combining the advantages of highly efficient enzymatic synthesis of sialoside building blocks, and
diverse chemical glycosylation, this chemoenzymatic approach is practical for obtaining complex sialos-
ides and their analogues.
Ó 2008 Elsevier Ltd. All rights reserved.
Dedicated to Professor Yongzheng Hui
on the occasion of his 70th birthday
Keywords:
Sialyl Lewis x
Sialoside
Oligosaccharide
Chemoenzymatic synthesis
1. Introduction
generate and regenerate sugar nucleotides. Chemical glycosylation
offers more diversity but is complicated by inherent multiple pro-
P-, E-, and L-selectins are C-type lectins that are involved in
lymphocyte homing, initial process in leukocyte recruitment to
inflammation sites, and cancer metathesis.1–6 Sialyl Lewis x (SLex)
and its sulfated derivatives in the presence or the absence of a pep-
tide carrier are the most common selectin ligands. The sialyl Lewis
tection and deprotection processes and difficulties in choosing
suitable protecting groups. For example, chemical sialylation on
the 3-OH of Gal usually results in low yield and a mixture of
a-
and b-linked sialoside products due to the sterically hindered ano-
meric position and the lack of a participating group on the C3 of
sialic acid.25–27 In addition, the low reactivity of the hydroxyl
groups in N-acetylglucosamine acceptor during glycosylation28,29
x
tetrasaccharide (a-Neup5Ac-(2?3)-b-Galp-(1?4)[a-Fucp-(1?
3)]-GlcpNAc-R) has been a leading structure for the development
of new anti-inflammatory agents.7 SLex is also a tumor-associated
antigen; it has been found on cancer cells and in the sera from
patients with breast, colon, lung, stomach, ovarian, pancreatic,
prostate, or urinary bladder cancer. Therefore, it has been a key
component for developing synthetic carbohydrate-based cancer
vaccines.8–10
and the lability of the
a-L
-Fuc linkage under acidic condition30
complicate the total chemical synthesis of SLex.
Recently, chemically synthesized sialylated oligosaccharide
building blocks have been successfully used in the assembly of
complex sialosides.31,32 These sialylated oligosaccharide donor
building blocks can be easily accessed by enzyme-catalyzed
approaches, which greatly simplify the overall synthesis of
complex sialosides such as SLex. Several groups have successfully
employed this method for synthesizing sialosides.33–35 We present
here the work showing that the SLex structure can be efficiently
obtained by chemical glycosylation using a p-tolyl thioglycosyl
donor formed by a sialyltransferase-catalyzed reaction.
The important biological activity of SLex has attracted much
interest in developing chemical, enzymatic, or chemoenzymatic
approaches for the synthesis of this oligosaccharide and its deriva-
tives.6,11–24 Despite recent advances, the synthesis of SLex is still
challenging.25–27 Enzymatic synthesis of SLex using a number of re-
combinant enzymes proceeds with regio- and stereoselectively,
but its application to large-scale synthesis is limited by the acces-
sibility of biosynthetic enzymes, the strict substrate specificity of
glycosyltransferases, and the involvement of multiple enzymes to
2. Results and discussion
Recently, our group has developed an efficient one-pot three-
enzyme chemoenzymatic approach for preparing natural and
non-natural sialosides from simple sialic acid precursors using
* Corresponding author. Tel.: +1 530 754 6037; fax: +1 530 752 8995.
E-mail address: chen@chem.ucdavis.edu (X. Chen).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.06.020

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H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
highly active and highly flexible recombinant bacterial sialoside
biosynthetic enzymes.36a,b,37 Due to the high expression level and
reactivity of these bacterial enzymes, the synthesis can be rou-
tinely carried out in large scale (>100 mg) and can give various
sialosides in high yields. In this chemoenzymatic method, chemical
synthesis of the substrate derivatives is applied prior to the en-
zyme-catalyzed sialylation reaction. An alternative chemoenzy-
matic approach is to carry out enzymatic sialylation reaction to
obtain sialoside building blocks before chemical glycosylation
reactions for the synthesis of more complex oligosaccharides. The
latter approach will be extremely useful, as a way to avoid the
strict substrate specificity of some glycosyltransferases, and to pro-
vide more diverse carbohydrate or glycoconjugate structures that
may not be accessible by enzymatic synthesis or the former che-
moenzymatic method. The latter chemoenzymatic approach also
avoids the difficulties in chemical construction of some glycosidic
bonds including sialyl linkages.
Several examples of applying the alternative chemoenzymatic
method in the synthesis of complex carbohydrates have been
reported. Hayashi et al. used a sialotrisaccharide building block
obtained from enzymatically synthesized oligosaccharide followed
by protection and selective deprotection as a glycosyl acceptor for
chemical fucosylation to provide SLex tetrasaccharide donors,
which can be used for further introduction of different aglycons.33
Similarly, Whitfield’s group used phenyl thioglycosides formed
from enzymatically assembled galactodisaccharide or sialotrisac-
charide as glycosyl donors for the synthesis of more complex oligo-
saccharides.35 In these examples, the b-Galp-(1?4)-GlcpNAc
moiety was assembled by a b-(1?4)-galactosyltransferase-cata-
lyzed reaction. We present here that a SLex tetrasaccharide with
a propylamine aglycon 1 (Fig. 1) can be obtained efficiently, with-
out using a galactosyltransferase, from a p-tolyl thioglycosyl donor
formed from a sialodisaccharide provided by a sialyltransferase-
catalyzed reaction. A similar aryl thioglycoside disaccharide donor
The synthesis of sialodisaccharide donor 2 started from commer-
cially available N-acetylmannosamine (ManNAc) and known thiog-
alactoside 540 by the one-pot three-enzyme approach described
previously.36a,37 In this enzymatic reaction, ManNAc is converted
by an Escherichia coli K-12 sialic acid aldolase to N-acetylneurami-
nic acid, which is activated by an Neisseria meningitidis CMP-sialic
acid synthetase before being transferred to p-tolyl thiogalatoside 5
by
a
multifunctional Pasteurella multocida sialyltransferase
(PmST1) (Scheme 1).36a,b Gram-scale synthesis of sialodisaccharide
6 was achieved in quantitative yield as shown by TLC analysis. The
reaction was stopped by adding an equal volume of ethanol and
the mixture was centrifuged to remove precipitates. The superna-
tant was concentrated by rotary evaporation and the sialoside 6
was converted to protected methyl ester 2 in a yield of 79% over
three steps by following a reported method.33
2-N-Phthaloyl protected glycoside 3, having a bulky silyl group
at O-6 and two free hydroxy groups at C3 and C4, was chosen as
the glycosylation acceptor for sialodisaccharide 2. The bulky
N-phthalimido protecting group in acceptor 3 provides steric
hindrance to the neighboring C-3 hydroxyl group and enhances
the reactivity of the C-4 hydroxyl group by masking the amide
group, which disrupts amide-involved intermolecular hydrogen
bonding.29 Acceptor 3 was prepared in 81% overall yield by glyco-
sylation of trichloroacetimidate donor 841 with 3-chloropropanol
followed by SN2 substitution of the chlorine by an azido group,
deacetylation, and selective silylation at O-6 with tert-butyl-
diphenylsilyl chloride (TBDPS-Cl) (Scheme 2). Thiofucosyl donor
4 was prepared by following a reported method.42
With building blocks 2–4 in hand, the next step was to explore
the linear assembly of SLex tetrasaccharide 1 (Scheme 3). After
screening different promoters and activation systems including
BSP–Tf2O43 and p-TolSCl–AgOTf44, only NIS–TfOH45,46 gave the sat-
isfactory yield in the chemoselective glycosylation of acceptor 3
using thiosialoside 2. The low yields resulting from BSP–Tf2O43 or
p-TolSCl–AgOTf44 under pre-activation conditions may be due to
the inherent low reactivity of the sialyl moiety in the glycosyl
donor.25 As shown in Scheme 3, using NIS–TfOH, glycosylation of
3 using 2 led to regioselective formation of a (1?4)-glycosidic
bond between the anomeric carbon on the Galp residue in sialod-
isaccharide donor 2 and the acceptor 3. The regioselectivity was
the result of the relatively low activity and the relatively large size
of sialodisaccharide donor 2, as well as the steric hindrance to
C3–OH and the enhanced reactivity of C4–OH caused by the bulky
N-phthalimido protecting group in acceptor 3.29 The stereoselec-
tive formation of the b-galactosyl linkage was the result of the
neighboring group participation of the C-2 acetylated OH on the
Gal in sialodisaccharide donor 2. Trisaccharide 11 was achieved
in 87% yield.
has been used by Whitfield’s group for synthesizing
a-(2?3)-
linked sialyl LacNAc derivatives but has not been shown for the
synthesis of SLex.34 The propylamine aglycon in the target com-
pound 1 can be used as a chemical handle for further conjugation
to facilitate biological studies.
Retrosynthetic analysis of the target molecule 1 indicates that it
can be chemically assembled from sialodisaccharide donor 2, thio-
fucosyl donor 4, and glucosamine acceptor 3 (Fig. 2).
Thioglycosides were chosen as donors due to their superior sta-
bility and their easy transferability to other glycosyl donors.38,39
HO
OH
OH
-
CO2
OH
O
OH
Subsequent
a-fucosylation of the free hydroxyl group at C3 of
O
O
O
AcHN
O
NH2
O
O
glucosamine in compound 11 using NIS–TfOH gave tetrasaccharide
12 in a yield of 92%. In this reaction, thiofucoside 4 was used in an
excess amount (2.5 equiv) to achieve a high yield. The efficient
fucosylation in this step was due to the smaller size and the higher
reactivity of thiofucosyl donor 4 compared to sialodisaccharide
donor 2. Removal of the tert-butyldiphenylsilyl and the 2-N-phthalyl
groups by HF–pyridine and N2H4ꢀH2O, respectively, followed by
HO
NHAc
HO
1: SLex-ProNH2
OH
O
OH
OH
HO
Figure 1. SLex tetrasaccharide with a propylamine aglycon.
OAc
OAc
AcO
OTBDPS
CO2Me
OAc
O
STol
OBn
O
O
HO
HO
STol
O
O
O
N3
AcHN
OBn
BnO
AcO
NPhth
AcO
OAc
2
3
4
Figure 2. Building blocks for the synthesis of SLex tetrasaccharide 1.

H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
2865
HO
-O2C
O
OH
O
HO
a
HO
OH
O
HO
HO
OH
HO
ManNAc
+
O
STol
STol
AcHN
OH
OH
5
6
O
MeO2C
OAc
OAc
OAc
OAc
AcO
b
AcO
AcO OAc
O
c
O
STol
O
O
O
O
STol
AcHN
AcHN
O
OAc
AcO
AcO
AcO OAc
7
2
Scheme 1. Reagents and conditions: (a) sodium pyruvate, CTP, MgCl2, Tris–HCl buffer (100 mM, pH 8.5), E. coli sialic acid aldolase, N. meningitidis CMP-sialic acid synthetase,
P. multocida sialyltransferase, quantitative; (b) Ac2O, pyridine, 91%; (c) (i) DMAP, MeOH, 90%; (ii) Ac2O, pyridine, 97%.
OAc
O
OAc
O
a
AcO
AcO
AcO
AcO
O
CCl3
NH
O
Cl
NPhth
NPhth
8
9
OTBDPS
OH
c
b
O
O
HO
HO
HO
HO
O
N3
O
NPhth
10
N3
NPhth
3
Scheme 2. Reagents and conditions: (a) 3-chloropropanol, 4 Å molecular sieves, CH2Cl2, TMSOTf, rt, 95%; (b) (i) NaN3, DMF, TBAI, 60 °C, 98%; (ii) NaOMe–MeOH, 100%; (c)
TBDPSCl, pyridine, DMAP, 60 °C, 87%.
OAc
OAc
AcO
CO2Me
O
OTBDPS
OAc
a
+
2
3
O
O
O
AcHN
O
N3
O
HO
AcO
NPhth
N3
AcO
OAc
11
OAc
OAc
AcO
CO2Me
O
OTBDPS
OAc
O
O
b
c
O
AcHN
O
O
O
AcO
NPhth
AcO
OAc
O
OBn
OBn
BnO
12
OAc
OAc
AcO
CO2Me
O
OAc
OAc
O
d
1
O
O
O
AcHN
O
N3
O
AcO
NHAc
AcO
OAc
O
OBn
OBn
BnO
13
Scheme 3. Reagents and conditions: (a) NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 0 °C to rt, 87%; (b) thiofucoside 4, NIS, TfOH, 4 Å molecular sieve, CH2Cl2, 0 °C to rt, 92%; (c) (i)
HF–pyridine, THF, pyridine, rt; (ii) N2H4ꢀH2O, MeOH, 80 °C; (iii) Ac2O, pyridine, 57% over three steps; (d) (i) 1,3-propanoldithiol, Et3N, pyridine, H2O, 50 °C; (ii) NaOMe–MeOH
then add H2O, rt; (iii) Pd(OH)2, H2, MeOH–H2O–AcOH, rt, BioGel P-2, 61% over three steps.
acetylation, the fully protected tetrasaccharide 13 was produced in
57% over three steps.
Because hydrogenation of benzyl groups in the presence of azi-
do group usually leads to low yield and complicated product mix-
tures, it was not used here. Instead, a two-step strategy was
applied. In the first step, the azido group in 13 was reduced
to an amino group using 1,3-propanoldithiol.47 After deacetylation
and saponification, benzyl groups were removed by hydrogena-
tion using Pd(OH)2 and H2 to give the desired SLex tetrasaccharide
1. After gel filtration purification, SLex tetrasaccharide 1 was

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H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
obtained in 61% yield over three steps of reduction and
deprotection.
4.3. p-Methylphenyl (5-N-acetyl-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosyl)-(2?3)-1-thio-b-D-
galactopyranoside (6)
3. Conclusion
The sialoside 6 was prepared using the one-pot three-enzyme
approach as reported.36a,37 ManNAc (464 mg, 2.1 mmol) was used
as sialic acid precursor and thiogalactopyranoside 5 (500 mg,
1.8 mmol) was used as sialyltransferase acceptor. The reaction
was carried out at room temperature (25 °C) in a total volume of
80 mL in the presence of CTP (1 equiv), sodium pyruvate (3 equiv),
MgCl2 (20 mM), Tris–HCl buffer (100 mM, pH 8.5), E. coli sialic acid
aldolase (5 mg), N. meningitidis CMP-sialic acid synthetase (1 mg),
and P. multocida sialyltransferase (PmST1, 1 mg). The reaction
was quenched by adding the same volume of ethanol when TLC
indicated the completion of the reaction. The reaction mixture
was centrifuged at 7000g for 30 min. to remove precipitates. The
supernatant was concentrated by rotary evaporation and lyophi-
lized to white solid which was used directly for acetylation in
the next step. Sialoside 6 was achieved in quantitative yield based
on TLC and purifying partial sialoside 6 by passing through a BioGel
P-2 gel filtration column. 1H NMR (600 MHz, D2O) d 7.34 (d, 2H,
J = 7.8 Hz), 7.11 (d, 2H, J = 7.2 Hz), 4.60 (d, 1H, J = 9.6 Hz), 3.98
(dd, 1H, J = 9.6, 3.6 Hz), 3.84 (d, 1H, J = 3.0 Hz), 3.73–3.68 (m, 3H),
3.57–3.44 (m, 8H), 2.61 (dd, 1H, J = 12.0, 4.8 Hz), 2.19 (s, 3H),
1.88 (s, 3H), 1.65 (t, 1H, J = 12.6 Hz); 13C NMR (150 MHz, D2O) d
175.16, 174.02, 138.96, 132.38, 130.06, 128.30, 100.07, 87.69,
78.89, 77.16, 73.01, 71.91, 68.50, 68.20, 67.88, 67.54, 62.60,
59.51, 51.81, 39.75, 22.18, 20.30; ESIMS: m/z calcd for
[C24H34NO13S]+: 576.1751, found: 576.1742.
In conclusion, an alternative chemoenzymatic approach was
used for efficient assembly of the biologically important SLex
tetrasaccharide. Applying highly efficient and selective enzymatic
synthesis to obtain oligosaccharide building blocks for diverse
chemical synthesis was proven to be highly valuable in obtaining
complex sialosides.
4. Experimental
4.1. General
Chemicals were purchased and used without further purifica-
tion. 1H NMR and 13C NMR spectra were recorded on Mercury-
300, Varian Inova-400, or Varian-600 spectrometer. Low and high
resolution ESI mass spectra were obtained at the Mass Spectro-
metry Facility in the University of California at Davis. Silica Gel
60 Å (200–425 mesh, Sorbent technologies) was used for flash
column chromatography. Thin-layer chromatography (TLC) was
performed on silica gel plates 60 GF254 (Sorbent technologies)
using anisaldehyde sugar stain or 5% sulfuric acid in ethanol stain
for detection. Gel filtration chromatography was performed using a
column (100 cm ꢁ 2.5 cm) packed with BioGel P-2 Fine resins (Bio-
Rad, Hercules, CA).
4.2. p-Methylphenyl 1-thio-a-D-galactopyranoside (5)
4.4. p-Methylphenyl [(5-N-acetyl-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
tone)]-(2?3)-4,6-di-O-acetyl-1-thio-b-
D-glycero-a-D
-galacto-2-nonulopyranosyl-10?2-lac-
BF3ꢀOEt2 (2.6 mL, 20.6 mmol) was added to a mixture of
1,2,3,4,6-penta-O-acetyl- -galactopyranose (3.0 g, 7.7 mmol) and
D
-galactopyranoside (7)
D
p-thiocresol (1.9 g, 15.5 mmol) in anhydrous CH2Cl2 (50 mL) at
0 °C. The mixture was stirred for 2 h at 0 °C before being allowed
to warm to room temperature followed by continuous stirring
overnight. The reaction mixture was diluted with CH2Cl2
(100 mL), washed with saturated NaHCO3 and then with brine. It
was then dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash chromato-
graphy on silica gel (1:2, EtOAc–hexane) to give p-methylphenyl
Crude thiosialoside 6 (1.2 g, 1.8 mmol, about 90% purity) was dis-
solved in pyridine (40 mL). To this solution at 0 °C was added Ac2O
(20 mL). The reaction mixture was allowed to warm to room tem-
perature and stirred overnight. After concentration by rotary evap-
oration, the residue was purified by flash chromatography on silica
gel (3:3:0.5 EtOAc–hexane–MeOH) to give lactone 7 as white solid
(1.47 g, 91% yield base on the amount of pure thioglycoside 6). 1H
NMR (600 MHz, CDCl3) d 7.39 (d, 2H, J = 7.8 Hz), 7.07 (d, 2H,
J = 7.8 Hz), 6.04 (d, 1H, J = 10.2 Hz), 5.46 (d, 1H, J = 3.0 Hz), 5.39
(dt, 1H, J = 10.8, 4.8 Hz), 5.29 (d, 1H, J = 5.4 Hz), 5.18 (dt, 1H,
J = 6.0, 2.4 Hz), 4.93 (t, 1H, J = 10.2 Hz), 4.58 (d, 1H, J = 9.6 Hz), 4.32
(dd, 1H, J = 12.6, 3.0 Hz), 4.19–4.13 (m, 2H), 4.09 (dd, 2H, J = 12.0,
6.6 Hz), 4.00 (dd, 1H, J = 10.2, 3.0 Hz), 3.86 (t, 1H, J = 6.0 Hz), 3.75
(d, 1H, J = 10.2 Hz), 2.47 (dd, 1H, J = 13.8, 6.0 Hz), 2.29, 2.22, 2.19,
2.07, 2.06, 2.05, 2.00, 1.98, 1.86 (s, 3H for each), 1.84 (t, 1H,
J = 8.4 Hz); 13C NMR (150 MHz, CDCl3) d 171.15, 171.11, 170.91,
170.79, 170.70, 170.62, 170.16, 169.95, 164.01, 138.85, 133.19,
130.04, 129.65, 129.23, 128.42, 97.35, 87.18, 75.71, 74.62, 73.39,
72.54, 70.51, 69.59, 67.82, 66.71, 62.80, 61.92, 49.17, 37.76, 23.32,
21.35, 21.23, 21.16, 21.09, 20.99, 20.85, 20.70; ESIMS: m/z calcd
for [C36H45NO18S+Na]+: 834.2255, found: 834.2292.
2,3,4,6-tetra-O-acetyl-1-thio-a-D-galactopyranoside (3.2 g, 92%)
as a syrup. 1H NMR (600 MHz, CDCl3) d 7.36 (d, 2H, J = 8.4 Hz),
7.07 (d, 2H, J = 8.4 Hz), 5.35 (d, 1H, J = 3.0 Hz), 5.16 (t, 1H,
J = 10.2 Hz), 4.99 (dd, 1H, J = 10.2, 3.6 Hz), 4.61 (d, 1H,
J = 10.2 Hz), 4.13 (dd, 1H, J = 11.4, 6.6 Hz), 4.06 (dd, 1H, J = 11.4,
6.6 Hz), 3.87 (t, 1H, J = 7.2 Hz), 2.29, 2.06, 2.04, 1.99, 1.92(s, 3H
for each); 13C NMR (150 MHz, CDCl3) d 170.54, 170.38, 170.22,
169.60, 138.60, 133.30, 129.83, 128.82, 87.03, 74.52, 72.20, 67.46,
67.43, 61.80, 21.35, 21.05, 20.86, 20.82, 20.77. The NMR data were
consistent with that reported.40
To a solution of p-methylphenyl 2,3,4,6-tetra-O-acetyl-1-thio-
a-D-galactopyranoside (3.2 g, 7.04 mmol) in dry MeOH (100 mL),
was added NaOMe (200 mg). After stirring for 2 h, the reaction
mixture was neutralized with Dowex 50W (H+), filtered, and con-
centrated to give the p-methylphenyl 1-thio-a-D-galactopyran-
4.5. p-Methylphenyl [methyl (5-N-acetyl-4,7,8,9-tetra-O-acetyl-
oside 540 as white solid (2.0 g, 100%). The compound was used
directly for the following enzyme reaction without any further
purification. 1H NMR (600 MHz, D2O) d 7.30 (d, 2H, J = 7.8 Hz),
7.07 (d, 2H, J = 7.8 Hz), 4.50 (d, 1H, J = 10.2 Hz), 3.79 (d, 1H,
J = 3.0 Hz), 3.58 (dd, 1H, J = 12.6, 9.0 Hz), 3.54–3.51 (m, 2H), 3.49
(dd, 1H, J = 9.0, 3.0 Hz), 3.42 (t, 1H, J = 9.6 Hz), 2.15 (s, 3H); 13C
NMR (150 MHz, D2O) d 138.84, 131.99, 130.10, 128.84, 88.55,
79.08, 74.08, 69.31, 68.80, 61.05, 20.25. The NMR data were consis-
tent with that reported.40
3,5-dideoxy-
(2?3)-2,4,6-tri-O-acetyl-1-thio-b-
D
-glycero-
a
-
D
-galacto-2-nonulopyranosyl)onate]-
D
-galactopyranoside (2)
To a solution of lactone 7 (1.4 g, 1.7 mmol) in MeOH (41 mL) at
0 °C, was added 4-(dimethylamino)pyridine (4.6 mL of 10% solu-
tion; 0.46 mmol). The reaction mixture was stirred for 24 h at room
temperature and concentrated under reduced pressure. The resi-
due was purified by flash chromatography on silica gel (3:3:0.5
hexane–EtOAc–MeOH) to give p-methylphenyl [methyl (5-N-acet-

H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
2867
yl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
ulopyranosyl) onate]-(2?3)-4,6-di-O-acetyl-1-thio-b-
D
-glycero-
a
-
D
-galacto-2-non-
-galacto-
(150 mL), washed with brine, dried over MgSO4, filtered and puri-
fied by flash chromatography (1:1 EtOAc–hexane) to give 3-azido-
D
pyranoside as a white solid (1.3 g, 90%). 1H NMR (600 MHz, CDCl3)
d 7.47 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 8.4 Hz), 5.42–5.39 (m, 1H),
5.34 (s, 1H), 5.31 (dd, 1H, J = 9.0, 2.4 Hz), 4.95–4.90 (m, 2H), 4.73 (d,
1H, J = 9.0 Hz), 4.39 (t, 1H, J = 4.2 Hz), 4.24 (dd, 1H, J = 12.0, 2.4 Hz),
4.04–3.97 (m, 4H), 3.83 (s, 3H), 3.82 (dd, 2H, J = 3.6, 2.4 Hz), 3.61 (t,
1H, J = 8.4 Hz), 2.63 (dd, 1H, J = 12.6, 4.2 Hz), 2.30, 2.11, 2.09, 2.02,
2.00, 1.99, 1.97, 1.84 (s, 3H for each), 1.85 (t, 1H, J = 12.6 Hz); 13C
NMR (150 MHz, CDCl3) d 171.00, 170.81, 170.64, 170.57, 170.45,
170.33, 170.19, 167.84, 137.88, 132.94, 129.65, 129.31, 97.17,
87.66, 75.35, 74.55, 72.61, 69.00, 68.13, 67.94, 66.92, 62.58,
62.46, 53.44, 49.49, 37.84, 23.36, 21.59, 21.33, 21.04, 21.00,
20.98, 20.94, 20.92, 20.80.
propyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyran-
oside (2.5 g, 98%) as white solid. 1H NMR (300 MHz, CDCl3) d
7.86 (dd, 2H, J = 6.0, 3.3 Hz), 7.74 (dd, 2H, J = 5.1, 2.7 Hz), 5.78
(dd, 1H, J = 10.8, 9.0 Hz), 5.37 (d, 1H, J = 8.4 Hz), 5.17 (dd, 1H,
J = 10.2, 9.0 Hz), 4.35–4.28 (m, 2H), 4.18 (dd, 1H, J = 12.3, 2.1 Hz),
3.94–3.84 (m, 2H), 3.58–3.51 (m, 1H), 3.24–3.10 (m, 2H), 2.11,
2.03, 1.86 (s, 3H for each), 1.81–1.63 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 170.98, 170.42, 169.73, 134.64, 123.89, 98.41, 72.15,
70.96, 69.17, 66.83, 62.22, 54.80, 48.16, 29.01, 21.01, 20.88,
20.70; ESIMS: m/z calcd for [C23H26N4O10+Na]+: 541.1547, found:
541.1529.
To a solution of 3-azidopropyl 3,4,6-tri-O-acetyl-2-deoxy-2-
To a solution of the methyl ester (1.3 g, 1.5 mmol) containing a
free C2–OH at Gal obtained from previous step in pyridine (30 mL)
at 0 °C, was added Ac2O (10 mL). The reaction mixture was stirred
overnight at room temperature before it was concentrated and the
residue was purified by flash chromatography (3:3:0.5 hexane–
EtOAc–MeOH) to give compound 2 as white solid (1.3 g, 97%). 1H
NMR (600 MHz, CDCl3) d 7.37 (d, 2H, J = 7.8 Hz), 7.05 (d, 2H,
J = 8.4 Hz), 5.34 (d, 1H, J = 10.2 Hz), 5.48 (dt, 1H, J = 2.4, 8.4 Hz),
5.32 (dd, 1H, J = 8.4, 2.4 Hz), 4.98 (t, 1H, J = 9.6 Hz), 4.88 (d, 1H,
J = 3.6 Hz), 4.80–4.84 (m, 1H), 4.76 (d, 1H, J = 10.2 Hz), 4.58 (dd,
1H, J = 9.6, 3.0 Hz), 4.33 (dd, 1H, J = 12.6, 3.0 Hz), 3.99–4.07 (m,
3H), 3.94 (dd, 1H, J = 12.6, 6.0 Hz), 3.85 (t, 1H, J = 7.2 Hz), 3.77 (s,
3H), 3.63 (dd, 1H, J = 10.2, 2.4 Hz), 2.27, 2.20, 2.10, 2.03, 2.02,
1.99, 1.95, 1.93, 1.80 (s, 3H for each), 1.63 (t, 1H, J = 12.6 Hz); 13C
NMR (75 MHz, CDCl3) d 170.95, 170.73, 170.67, 170.59, 170.45,
169.88, 169.86, 168.20, 138.03, 132.91, 129.66, 129.27, 96.96,
86.09, 74.37, 72.44, 72.28, 69.53, 68.17, 68.01, 67.37, 62.69,
62.47, 53.33, 49.12, 37.75, 24.20, 23.30, 21.65, 21.30, 21.19,
20.96, 20.89, 20.84; ESIMS: m/z calcd for [C39H51NO20S+Na]+:
908.2623, found: 908.2635.
phthalimido-b-D-glucopyranoside (2.5 g, 4.8 mmol) in MeOH
(100 mL) was added NaOMe (200 mg). After stirring for 2 h at room
temperature, the reaction mixture was neutralized with Dowex
50W (H+), filtered and concentrated under reduced pressure. The
residue was purified by flash chromatography (10:1 CH2Cl2–
MeOH) to give glycoside 10 (1.9 g, 100%) as white solid. 1H NMR
(600 MHz, D2O) d 7.86 (dd, 2H, J = 4.8, 3.0 Hz), 7.81 (dd, 2H,
J = 5.4, 3.0 Hz), 5.16 (d, 1H, J = 8.4 Hz), 4.26 (dd, 1H, J = 10.2, 7.8
Hz), 3.97 (dd, 1H, J = 10.2, 8.4 Hz), 3.94–3.88 (m, 2H), 3.74 (dd,
1H, J = 12.0, 5.4 Hz), 3.53–3.49 (m, 1H), 3.45–3.38 (m, 2H), 3.22
(t, 1H, J = 8.4 Hz), 3.14–3.09 (m, 2H), 1.69–1.62 (m, 2H), 1.00 (t,
2H, J = 7.2 Hz); 13C NMR (75 MHz, CDCl3)
d 168.72, 134.43,
131.81, 98.62, 75.79, 71.79, 71.54, 66.47, 61.93, 56.84, 48.17,
28.99; ESIMS: m/z calcd for [C17H20N4O7+Na]+: 415.1230, found:
415.1228.
4.8. 3-Azidopropyl 6-tert-O-butyldiphenylsilyl-2-deoxy-2-
phthalimido-b-D-glucopyranoside (3)
To a solution of glycoside 10 (1.8 g, 4.6 mmol) in dry pyridine
(34 mL), were added TBDPSCl (2.4 mL, mmol) and DMAP
(100 mg). The reaction mixture was stirred overnight at 60 °C
before it was concentrated and purified by flash chromatography
(20:1 CH2Cl2–MeOH) to give compound 3 as syrup (2.5 g, 87%).
1H NMR (600 MHz, CDCl3) d 7.81 (dd, 2H, J = 6.0, 3.6 Hz), 7.70–
7.68 (m, 6H), 7.45–7.38 (m, 6H), 5.18 (d, 1H, J = 8.4 Hz), 4.34 (dd,
1H, J = 10.8, 8.4 Hz), 4.09 (dd, 1H, J = 10.8, 9.0 Hz), 3.94 (dd, 2H,
J = 4.8, 1.8 Hz), 3.81–3.78 (m, 1H), 3.66 (t, 1H, J = 9.0 Hz), 3.59–
3.56 (m, 1H), 3.47–3.44 (m, 1H), 3.12 (dt, 2H, J = 6.0, 1.8 Hz),
1.73–1.60 (m, 2H), 1.06 (s, 9H); 13C NMR (150 MHz, CDCl3) d
168.67, 135.88, 135.82, 134.42, 131.84, 130.21, 128.12, 128.09,
123.70, 98.38, 74.64, 74.50, 71.93, 66.17, 65.24, 56.46, 48.27,
29.08, 27.03, 19.44; ESIMS: m/z calcd for [C33H38N4O7Si+Na]+:
653.2408, found: 653.2398.
4.6. 3-Chloropropyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-
b-
D-glucopyranoside (9)
A mixture of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-D-glu-
copyranosyl imidate (1.8 g, 3.1 mmol), 3-chloropropanol
(0.52 mL, 6.2 mmol), and 4 Å molecular sieves (2.0 g) in dry CH2Cl2
(50 mL) was stirred at room temperature for 30 min. The reaction
mixture was cooled down to ꢂ78 °C and TMSOTf (20
lL,
0.11 mmol) was added. After 1 h, the reaction mixture was allowed
to warm to room temperature and stirred for another 2 h when the
reaction was completed as indicated by TLC. Et3N (0.1 mL) was
added and the reaction mixture was filtered and concentrated.
The residue was purified by flash chromatography (1:1 EtOAc–hex-
ane) on silica gel to give the glycoside 9 as solid foam (1.51 g, 95%).
1H NMR (300 MHz, CDCl3) d 7.85 (dd, 2H, J = 5.7, 3.0 Hz), 7.74 (dd,
2H, J = 5.7, 2.7 Hz), 5.80 (dd, 1H, J = 10.5, 9.0 Hz), 5.37 (d, 1H,
J = 8.7 Hz), 5.16 (dd, 1H, J = 9.9, 9.0 Hz), 4.36–4.27 (m, 2H), 4.17
(dd, 1H, J = 12.3, 2.4 Hz), 3.98–3.85 (m, 2H), 3.67–3.59 (m, 1H),
3.44–3.30 (m, 2H), 2.11, 2.03, 1.86 (s, 3H for each), 1.96–1.78 (m,
2H); 13C NMR (75 MHz, CDCl3) d 170.89, 170.31, 169.64, 134.50,
131.50, 123.77, 98.50, 72.03, 70.84, 69.10, 66.63, 62.16, 54.74,
41.35, 32.19, 20.93, 20.79, 20.62; ESIMS: m/z calcd for
[C23H26ClNO10+Na]+: 534.1143, found: 534.1109.
4.9. p-Methylphenyl 2,3,4-tri-O-benzyl-1-thio-a-L-
fucopyranoside (4)
Compound
4 was prepared from L-fucose in 71% yield
in four steps by following procedures as reported previously.42
1H NMR (600 MHz, CDCl3): d 7.49 (d, 2H, J = 7.8 Hz), 7.41–7.29
(m, 15H), 7.02 (d, 2H, J = 7.8 Hz), 5.01 (d, 1H, J = 12.0 Hz), 4.81 (d,
1H, J = 10.2 Hz), 4.76–4.72 (m, 3H), 4.67 (d, 1H, J = 12.0 Hz), 4.55
(d, 1H, J = 10.2 Hz), 3.90 (t, 1H, J = 9.6 Hz), 3.64 (d, 1H, J = 3.0 Hz),
3.59 (dd, 1H, J = 9.6, 3.0 Hz), 3.51 (dd, 1H, J = 12.6, 6.0 Hz),
2.30 (s, 3H), 1.27 (d, 3H, J = 6.6 Hz); 13C NMR (75 MHz, CDCl3): d
139.00, 138.70, 138.62, 137.35, 132.42, 130.67, 129.76, 128.69,
128.61, 128.58, 128.39, 128.20, 127.95, 127.82, 127.69, 88.11,
84.81, 77.38, 76.80, 75.80, 74.80, 73.08, 21.38, 17.57. The 1H and
13C NMR spectra were consistent with those reported.42
4.7. 3-Azidopropyl 2-deoxy-2-phthalimido-b-D-
glucopyranoside (10)
To a solution of 9 (2.5 g, 4.9 mmol) in dry DMF (50 mL) were
added NaN3 (3.2 g, 48.8 mmol) and TBAI (100 mg). After stirring
overnight at 60 °C, the reaction mixture was diluted with EtOAc

2868
H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
4.10. 3-Azidopropyl [methyl (5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy- -glycero- -galacto-
2-nonulopyranonate)]-(2?3)-2,4,6-tri-O-acetyl-b-
galactopyranosyl-(1?4)-6-O-tert-butyldiphenylsilyl-2-
deoxy-2-phthalimido-b- -glucopyranoside (11)
1.08 (s, 9H); 13C NMR (150 MHz, CDCl3):
d 171.23, 170.86,
D
a-D
170.50, 170.24, 170.18, 169.83, 169.32, 168.12, 139.17, 138.99,
138.52, 136.28, 135.87, 134.38, 134.35, 133.27, 129.92, 129.87,
128.33, 128.27, 128.19, 128.10, 127.78, 127.76, 127.56, 127.37,
127.28, 127.17, 123.69, 99.69, 97.94, 97.60, 97.31, 79.84, 77.78,
76.64, 75.68, 75.15, 74.49, 73.24, 72.89, 72.71, 72.43, 71.79,
70.91, 70.73, 69.71, 68.80, 67.99, 67.37, 66.81, 65.63, 63.27,
62.08, 61.63, 60.66, 56.54, 53.70, 53.31, 49.32, 37.47, 28.94,
27.21, 23.33, 21.41, 21.27, 21.10, 21.05, 20.92, 20.91, 20.85,
20.72, 19.78, 16.95; ESIMS: m/z calcd for [C92H109N5O31Si+Na]+:
1830.6774, found: 1830.6538.
D
-
D
A mixture of sialoside donor 2 (360 mg, 0.41 mmol), glucosa-
mine acceptor 3 (216 mg, 0.34 mmol), and 4 Å molecular sieve
(300 mg) in dry CH2Cl2 was stirred for 30 min at room tempera-
ture. The mixture was cooled down to 0 °C and N-iodosuccinimide
(NIS, 185 mg, 0.82 mmol) and triflic acid (11 lL, 0.12 mmol) were
added. After stirring for 1 h at 0 °C, the reaction mixture was
warmed to room temperature and stirred for another 2 h when
TLC indicated the completion of the reaction. The reaction mixture
was neutralized by Et3N (0.2 mL), filtered, and concentrated under
reduce pressure. The residue was purified by flash chromatography
(3:3:0.5 EtOAc–hexane–MeOH) to give trisaccharide 11 as white
solid (415 mg, 87%). 1H NMR (600 MHz, CDCl3): d 7.80–7.79 (m,
2H), 7.68–7.66 (m, 6H), 7.37–7.30 (m, 6H), 5.72 (d, 1H,
J = 10.2 Hz), 5.35 (t, 1H, J = 8.4), 5.24 (d, 1H, J = 8.4 Hz), 5.16 (d,
1H, J = 8.4 Hz), 4.91 (t, 1H, J = 8.4 Hz), 4.78–4.74 (m, 2H), 4.61 (d,
1H, J = 9.6 Hz), 4.53–4.51 (m, 2H), 4.28 (dd, 1H, J = 10.2, 8.4 Hz),
4.20 (d, 1H, J = 12.6 Hz), 4.07–4.01 (m, 1H), 3.99–3.87 (m, 4H),
3.86–3.77 (m, 3H), 3.71 (s, 3H), 3.69–3.66 (m, 1H), 3.64 (t, 1H,
J = 9.6 Hz), 3.56 (d, 1H, J = 10.8 Hz), 3.45 (t, 1H, J = 8.4 Hz), 3.36–
3.32 (m, 1H), 3.08–3.01 (m, 2H), 2.46 (dd, 1H, J = 12.6, 4.2 Hz),
2.04, 1.99, 1.94, 1.91, 1.89, 1.76, 1.69, 1.65 (s, 3H for each), 1.63–
1.56 (m, 2H), 1.53 (t, 1H, J = 12.0 Hz), 0.99 (s, 9H); 13C NMR
4.12. 3-Azidopropyl [methyl (5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-
2-nonulopyranonate)]-(2?3)-2,4,6-tri-O-acetyl-b-
galactopyranosyl-(1?4)[(1?3)-2,3,4-tri-O-benzyl-
D-glycero-a-D-galacto-
D
-
a-L-
fucopyranosyl]-6-O-acetyl-2-deoxy-2-N-acetyl-b-D-
glucopyranoside (13)
To the solution of tetrasaccharide 12 (490 mg, 0.27 mmol) in
pyridine (0.6 mL) and THF (2.4 mL) at 0 °C, was added HF–pyridine
(65–70%, 0.26 mL). The reaction mixture was stirred overnight at
room temperature when TLC indicated the completion of the reac-
tion. The reaction mixture was cooled down to 0 °C and neutralized
using saturated NaHCO3 (10.0 mL). It was then extracted with
EtOAc and the combined organic phase was dried with Na2SO4, fil-
tered, and concentrated to give crude selectively deprotected tetra-
saccharide as white solid (480 mg). The white solid residue was
dissolved in MeOH (30.0 mL) and N2H4ꢀH2O (6.0 mL) was added.
The reaction mixture was heated at reflux at 80 °C for 10 h when
TLC indicated the completion of the reaction. The reaction mixture
was concentrated and co-evaporated with toluene. The solid resi-
due was dissolved in dry pyridine (20.0 mL) and Ac2O (5.0 mL)
was added at 0 °C. It was allowed to warm to room temperature
and stirred for overnight. The reaction mixture was concentrated
and purified by flash chromatography (3:3:0.5 EtOAc–hexane–
MeOH) to give compound 13 as white solid (235 mg, 57% for three
steps). 1H NMR (600 MHz, CDCl3): d 7.37–7.34 (m, 4H), 7.30–7.21
(m, 11H), 5.95 (d, 1H, J = 7.8 Hz), 5.63 (dt, 1H, J = 3.0 Hz), 5.43 (dd,
1H, J = 9.6, 2.4 Hz), 5.12 (d, 1H, J = 10.2 Hz), 5.04 (d, 1H, J = 3.6 Hz),
4.92 (d, 2H, J = 12.0 Hz), 4.83 (d, 1H, J = 12.0 Hz), 4.79–4.61 (m,
7H), 4.57 (d, 1H, J = 11.4, 3.6 Hz), 4.53 (d, 1H, J = 10.2, 3.0 Hz), 4.26
(d, 1H, J = 12.6, 3.0 Hz), 4.20 (dd, 1H, J = 12.0, 5.4 Hz), 4.14 (d, 1H,
J = 10.8 Hz), 4.12–4.06 (m, 3H), 3.95 (dd, 1H, J = 10.8, 2.4 Hz), 3.94
(d, 1H, J = 3.6 Hz), 3.87 (d, 2H, J = 7.8 Hz), 3.85 (dd, 1H, J = 11.4,
3.6 Hz), 3.82–3.79 (m, 2H), 3.69 (t, 2H, J = 7.2 Hz), 3.65 (s, 3H),
3.46–3.43 (m, 1H), 3.26–3.21 (m, 2H), 2.69 (dd, 1H, J = 13.2,
4.8 Hz), 2.23, 2.22, 2.06, 2.03, 2.02, 1.98, 1.92, 1.84, 1.81, 1.76 (s,
3H for each), 1.73–1.66 (m, 2H), 1.12 (d, 3H, J = 6.6 Hz); 13C NMR
(75 MHz, CDCl3):
d 170.88, 170.66, 170.60, 170.58, 170.39,
169.78, 169.69, 168.14, 136.00, 135.86, 134.33, 134.12, 133.51,
131.83, 129.92, 129.77, 127.88, 127.81, 101.61, 98.02, 96.93,
83.64, 75.43, 72.34, 71.33, 71.07, 70.61, 69.83, 69.49, 68.26,
67.36, 67.29, 65.98, 63.48, 62.62, 62.49, 60.61, 56.12, 53.37,
48.96, 37.70, 28.98, 23.21, 21.59, 21.22, 20.98, 20.93, 20.74,
20.52, 20.24, 19.47; ESIMS: m/z calcd for [C65H81N5O27Si+H]+:
1392.4966, found: 1392.4893.
4.11. 3-Azidopropyl [methyl (5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-
2-nonulopyranonate)]-(2?3)-2,4,6-tri-O-acetyl-b-
galactopyranosyl-(1?4)[(1?3)-2,3,4-tri-O-benzyl-
fucopyranosyl]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-
phthalimido-b- -glucopyranoside (12)
D-glycero-a-D-galacto-
D
-
a-L-
D
The mixture of thiofucosyl donor 4 (398 mg, 0.74 mmol), trisac-
charide acceptor 11 (410 mg, 0.29 mmol) and 4 Å molecular sieve
(400 mg) in dry CH2Cl2 was stirred for 30 min at room tempera-
ture. The mixture was cooled down to 0 °C, and N-iodosuccinimide
(NIS, 333 mg, 1.5 mmol) and triflic acid (20 lL, 0.23 mmol) were
added. After stirring for 1 h at 0 °C, the reaction mixture was
warmed to room temperature and stirred for another 3 h when
TLC indicated the completion of the reaction. The reaction mixture
was neutralized by Et3N (0.4 mL), filtered, and concentrated. The
residue was purified by flash chromatography (3:3:0.4 EtOAc–hex-
ane–MeOH) to give tetrasaccharide 12 as white solid (490 mg,
92%). 1H NMR (600 MHz, CDCl3): d 7.75–7.64 (m, 8H), 7.40–7.34
(m, 6H), 7.25–7.09 (m, 13H), 7.02 (d, 2H, J = 6.6 Hz), 5.43 (d, 1H,
J = 10.2 Hz), 5.37–5.34 (m, 2H), 4.99–4.88 (m, 5H), 4.76 (d, 1H,
J = 11.4 Hz), 4.71 (dd, 1H, J = 10.8, 9.0 Hz), 4.56–4.49 (m, 5H), 4.42
(d, 1H, J = 12.6 Hz), 4.29–4.25 (m, 2H), 4.16–4.08 (m, 4H), 4.00–
3.94 (m, 3H), 3.91–3.83 (m, 3H), 3.78 (s, 3H), 3.77 (dd, 1H,
J = 10.2, 2.4 Hz), 3.62 (d, 1H, J = 2.4 Hz), 3.60 (s, 1H), 3.57 (dd, 1H,
J = 10.2, 3.6 Hz), 3.42 (dt, 1H, J = 6.0 Hz), 3.11–3.07 (m, 1H), 2.93
(ddd, 1H, J = 2.4, 1.2, 1.2 Hz), 2.48 (dd, 1H, J = 12.6, 4.8 Hz), 2.09,
2.05, 2.02, 2.01, 1.99, 1.95, 1.92, 1.84, 1.76 (s, 3H for each), 1.71
(t, 1H, J = 12.6 Hz), 1.53–1.41 (m, 2H), 1.21 (d, 3H, J = 7.2 Hz),
(150 MHz, CDCl3): d 171.02, 171.00, 170.88, 170.66, 170.58,
170.38, 170.18, 170.08, 169.76, 169.63, 166.35, 161.67, 139.01,
138.99, 138.78, 128.69, 128.60, 128.54, 128.42, 128.08, 127.91,
127.80, 127.59, 127.44, 100.00, 99.79, 97.72, 95.24, 79.93, 77.25,
76.60, 74.61, 74.52, 73.83, 73.55, 72.85, 72.80, 70.86, 70.48, 69.54,
69.10, 67.39, 67.15, 66.90, 66.77, 66.25, 63.14, 62.24, 60.58, 49.07,
48.33, 38.41, 29.06, 23.39, 23.34, 22.21, 21.07, 21.05, 20.99, 20.97,
20.96, 20.80, 20.66, 16.94 ESIMS: m/z calcd for [C72H95N5O32+Na]+:
1564.5858, found: 1564.6082.
4.13. 3-Aminopropyl (5-acetamido-3,5-dideoxy-
-galacto-2-nonulopyranosylonic acid)-(2?3)-b-
galactopyranosyl-(1?4)[(1?3)- -fucopyranosyl]-2-
acetamido-2-deoxy-b- -glucopyranoside (1)
D-glycero-
a-D
D-
a-L
D
Tetrasaccharide 13 (230 mg, 0.15 mmol) was dissolved in the
mixture of pyridine (15 mL) and H2O (3 mL), then 1,3-propanol-

H. Cao et al. / Carbohydrate Research 343 (2008) 2863–2869
10. Seeberger, P. H.; Werz, D. B. Nature 2007, 446, 1046–1051.
2869
dithiol (0.55 mL) and Et3N (0.56 mL) were added. The reaction
mixture was stirred at 50 °C for 24 h when TLC indicated the
completion of the reaction. The mixture was concentrated and
co-evaporated with 5:1 toluene–ethanol, and purified by flash
chromatography (8:1 CH2Cl2–MeOH) to give amine intermediate
as white solid (224 mg). The amine intermediate (224 mg) was dis-
solved in dry MeOH (10 mL), and NaOMe (540 mg) was added.
After stirring for 10 h, H2O (10 mL) was added and the mixture
was stirred for another 4 h at room temperature. The reaction mix-
ture was neutralized with Dowex H+ resin, filtered, and concen-
trated. The white solid residue (163 mg) was dissolved in 5:5:1
MeOH–H2O–AcOH, and Pd(OH)2 (120 mg) was added. The mixture
was stirred at room temperature under atmospheric pressure H2
for 24 h. The mixture was filtered through Celite and concentrated.
The residue was purified by BioGel P-2 to give the targeted com-
pound 1 as white solid (83 mg, 61% for three steps). 1H NMR
(600 MHz, D2O): d 4.92 (d, 1H, J = 4.0 Hz), 4.61 (m, 1H), 4.34 (d,
2H, J = 8.4 Hz), 3.90 (dd, 1H, J = 9.6, 2.4 Hz), 3.84–3.81 (m, 2H),
3.78–3.65 (m, 9H), 3.59 (d, 1H, J = 3.0 Hz), 3.53–3.39 (m, 10H),
3.35 (dd, 1H, J = 10.8, 9.6 Hz), 2.90 (t, 2H, J = 7.2 Hz), 2.58 (dd, 1H,
J = 11.4, 4.2 Hz), 1.86 (s, 3H), 1.84 (s, 3H), 1.82–1.79 (m, 2H), 1.61
(t, 1H, J = 12.6 Hz), 0.98 (d, 3H, J = 6.0 Hz); 13C NMR (75 MHz,
CDCl3): d 175.21, 174.52, 101.52, 101.14, 99.68, 98.68, 75.48,
75.27, 74.78, 73.91, 73.38, 72.00, 71.37, 69.39, 69.29, 68.13,
67.95, 67.71, 67.29, 66.77, 63.06, 61.40, 59.72, 55.92, 51.65,
38.06, 37.71, 23.39, 22.31, 15.39; ESIMS: m/z calcd for
[C34H58N3O23]+: 876.3461, found: 876.3455.
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Acknowledgment
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This work was supported by NIH Grant U01CA128442.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.06.020.
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