Convergent synthesis of the tetrasaccharide repeating unit corresponding to the O-antigen of the verotoxin-producing Escherichia coli O176

Article abstract of DOI:10.1007/s10719-011-9351-4

A convergent synthesis of the tetrasaccharide repeating unit of the O-antigen of the verotoxin producing E. coli O176 has been achieved in excellent yield adopting a [2+2] block glycosylation strategy. The β-D-mannosidic moiety of the tetrasaccharide was prepared from β-Dglucoside and a-D-galactosamine moiety was derived from D-galactal. The tetrasaccharide was synthesized as its 2-trimethylsilylethyl glycoside in excellent yield. All intermediate steps are high yielding. The Author(s) 2011.

Full text of DOI:10.1007/s10719-011-9351-4

Glycoconj J (2011) 28:519–524
DOI 10.1007/s10719-011-9351-4
Convergent synthesis of the tetrasaccharide repeating unit
corresponding to the O-antigen of the verotoxin-producing
Escherichia coli O176
Goutam Guchhait & Anup Kumar Misra
Received: 22 July 2011 /Revised: 2 September 2011 /Accepted: 5 September 2011 /Published online: 20 September 2011
#
Springer Science+Business Media, LLC 2011
Abstract A convergent synthesis of the tetrasaccharide
repeating unit of the O-antigen of the verotoxin producing
E. coli O176 has been achieved in excellent yield adopting
a [2+2] block glycosylation strategy. The β-D-mannosidic
moiety of the tetrasaccharide was prepared from β-D-
glucoside and α-D-galactosamine moiety was derived from
D-galactal. The tetrasaccharide was synthesized as its 2-
trimethylsilylethyl glycoside in excellent yield. All inter-
mediate steps are high yielding.
haemolytic uraemic syndrome and termed as enterohaemor-
rhagic E. coli (EHEC) [4]. The VTEC strains are mostly
present in cattle and transmitted to humans by contaminated
food and water. The best characterized VTEC serotype is E.
coli O157:H7, which is responsible for fatal intestinal
disorders of humans in Europe, America and Japan [5, 6].
Besides, E. coli O157:H7, several other VTEC strains have
also been well characterized during last few years [7].
Recently, Widmalm et al. reported the tetrasaccharide
structure of the repeating unit of the O-antigenic polysac-
charide of the verotoxin-producing E. coli O176, which
contains a β-D-mannose and a α-D-galactosamine moiety
(Fig. 1) [8]. Cell wall O-antigenic polysaccharides are
attractive targets for the development of carbohydrate based
therapeutics, which is reflected in several reports in the
recent past [9, 10]. For a detailed understanding of the role
of O-antigenic polysaccharide of E. coli O176 in the
pathogenicity, a large quantity of the tetrasaccharide is
required for its use in several immunochemical experi-
ments. Since the natural source can not provide the required
amount of the tetrasaccharide, it is essential to develop a
concise chemical synthetic strategy for its preparation. We
report herein a convergent synthesis of the tetrasaccharide
repeating unit of the O-antigenic polysaccharide of E. coli
O176 as its 2-trimethylsilylethyl glycoside using block
synthetic strategy (Fig. 2). 2-Trimethylsilylethyl group (SE)
has been used as a temporary anomeric protecting group,
which could be removed under acidic condition to furnish
tetrasaccharide hemiacetal derivative [11].
.
.
.
Keywords E. coli Oligosaccharide Glycosylation
.
O-antigen Diarrhea
Introduction
Escherichia coli (E. coli), a Gram-negative, facultative
anaerobic pathogen can be predominantly found in the
gastrointestinal tract of humans [1]. Although, most of the
E. coli strains found in the gut flora are harmless, a certain
species of E. coli acquired virulence factors and causes
severe intestinal and urinary infections in humans and
animals. Most frequently encountered E. coli infections are
(a) diarrhea, (b) urinary tract infections and (c) meningitis
[2]. Verocytotoxin producing E. coli strains (VTEC) are
mostly responsible for several diarrheal outbreaks in the
developed and developing countries [3]. The VTEC strains
are also associated with the diarrhoeal diseases with life
threatening complications e.g. haemorrhagic colitis and
:
G. Guchhait A. K. Misra (*)
Results and discussion
Molecular Medicine Division, Bose Institute,
P-1/12, C.I.T. Scheme VII M,
Kolkata 700054, India
The convergent synthesis of the target tetrasaccharide 1 as
e-mail: akmisra69@gmail.com
its 2-trimethylsilylethyl glycoside was achieved using a

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Glycoconj J (2011) 28:519–524
→3)-α-D-GalpNAc-(1→4)-α-D-Manp-(1→2)-α-D-Manp-(1→2)-β-D-Manp-(1→
another experiment, HClO4-SiO2 promoted stereoselective
glycosylation [12] of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-
D-galactopyranosyl trichloroacetimidate (4) [17] and thio-
glycoside acceptor, ethyl 2,3-di-O-acetyl-6-O-benzyl-1-
thio-α-D-mannopyranoside (5) [18] furnished ethyl (3,4,6-
tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-
(1→4)-2,3-di-O-acetyl-6-O-benzyl-1-thio-α-D-mannopyra-
noside (8) in 78% yield. Presence of signals in the NMR
spectra of compound 8 confirmed its formation [δ 5.28 (br
Fig. 1 Structure of the tetrasaccharide repeating unit of the O-antigen
of the verotoxin-producing Escherichia coli O176
stereoselective [2+2] block glycosylation strategy. The
tetrasaccharide contains synthetically challenging β-D-
mannose and α-D-galactosamine moieties. The β-D-
mannoside acceptor (2) was prepared from a D-glucose
derivative using oxidation-reduction at the C-2 position.
The D-galactosamine donor (4) for the construction of α-D-
galactosamine moiety has been constructed from a D-
galactal derivative. The key features of the synthetic
strategy are: (a) stereoselective [2+2] block glycosylation;
(b) application of perchloric acid supported over silica
(HClO4-SiO2) as a solid acid catalyst in the glycosylation
of trichloroacetimidate derivative [12] and thioglycoside
[13] as well as in N- and O-acetylation [14]; (c) use of D-
galactal as a precursor of α-D-galactosamine moiety (4);
(d) activation of glycosyl trichloroacetimidate derivative (4)
in the presence of thioglycoside (5) tuning the orthogonal
property of anomeric thioethyl group of compound 5 [15];
(e) use of 2-trimethylsilylethyl group as temporary anome-
ric protecting group etc.
1
s, H-1D), 5.24 (br s, H-1C) in the H NMR and δ 99.0 (C-
1D), 81.9 (C-1C) in the 13C NMR spectra]. Stereoselective
glycosylation of disaccharide thioglycoside donor (8) with
disaccharide acceptor (7) in the presence of a combination
of NIS and HClO4-SiO2 [13] furnished 2-trimethylsilylethyl
(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyrano-
syl)-(1→4)-(2,3-di-O-acetyl-6-O-benzyl-α-D-mannopyra-
nosyl)-(1→2)-(3-O-benzyl-4,6-O-benzylidene-α-D-manno-
pyranosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-β-D-
mannopyranoside (9) in 77% yield. Spectral analysis of
compound 9 unambiguously confirmed its stereoselective
formation [δ 5.66 (s, PhCH), 5.57 (s, PHCH), 5.33 (br s, H-
1B), 5.31 (d, J=2.4 Hz, H-1D), 5.10 (br s, H-1C), 4.43 (br s,
1
H-1A) in the H NMR and δ 101.4 (PhCH), 101.3 (PhCH),
2-Trimethylsilylethyl 3-O-benzyl-4,6-O-benzylidene-β-
D-mannopyranoside (2) [11], derived from D-glucose in
seven steps was allowed to stereoselectively couple with
ethyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-1-thio-α-
D-mannopyranoside (3) [16] in the presence of a combina-
tion of N-iodosuccinimide (NIS) and HClO4-SiO2 [13] to
furnish 2-trimethylsilylethyl (2-O-acetyl-3-O-benzyl-4,6-O-
benzylidene-α-D-mannopyranosyl)-(1→2)-3-O-benzyl-4,6-
O-benzylidene-β-D-mannopyranoside (6) in 87% yield.
Presence of signals in the NMR spectra of compound 6
confirmed its formation [δ 5.68 (s, PhCH), 5.61 (s, PhCH),
100.5 (C-1D), 100.2 (C-1A), 99.2 (C-1C), 98.5 (C-1B) in the
13C NMR spectra]. Tetrasaccharide derivative 9 was
subjected to a sequence of reactions involving (a) hydro-
genolysis over Pearlman’s catalyst [19] for the reduction of
azido group and removal of O-benzyl ether and 4,6-O-
benzylidene acetal; (b) N- and O-acetylation using acetic
anhydride and HClO4-SiO2 [14] and (c) removal of O-
acetyl groups using sodium methoxide to give target
tetrasaccharide 1 as its 2-trimethylsilylethyl glycoside in
63% over all yield. The structure of the tetrasaccharide 1
was unambiguously confirmed using spectral analysis [δ
5.32 (br s, H-1B), 5.17 (d, J=3.6 Hz, H-1D), 5.00 (br s, H-
1
5.27 (br s, H-1B), 4.53 (br s, H-1A) in the H NMR and δ
1
101.6 (PhCH), 101.5 (PhCH), 100.2 (C-1A), 99.9 (C-1B) in
the 13C NMR spectra]. Deacetylation of compound 6 using
sodium methoxide in methanol furnished the disaccharide
acceptor 2-trimethylsilylethyl (3-O-benzyl-4,6-O-benzyli-
dene-α-D-mannopyranosyl)-(1→2)-3-O-benzyl-4,6-O-ben-
zylidene-β-D-mannopyranoside (7) in quantitative yield. In
1C), 4.52 (br s, H-1A) in the H NMR and δ 103.5 (C-1C),
101.5 (C-1B), 101.1 (2 C, C-1A, C-1D) in the 13C NMR
spectra] (Scheme 1). In order to establish the potential of 2-
trimethylsilylethyl group as a temporary anomeric protect-
ing group, compound 1 was subjected to a two step reaction
sequence involving (a) per-O-acetylation using acetic
Fig. 2 Chemical structure of the
synthesized tetrasaccharide as its
2-trimethylsilylethyl glycoside
(1)
HO
OH
OAc
O
Ph
O
O
HO
Ph
D
O
HO
O
HO
O
HO
HO
O
B
A
BnO
AcHN
O
OSE
O
BnO
C
SEt
SEt
2
3
O
HO
HO
HO
AcO
OAc
O
OAc
O
BnO
HO
AcO
B
O
NH
D
AcO
C
O
HO
HO
HO
N
O
3 O
CCl3
A
OSE
5
4
1
SE: 2-Trimethylsilylethyl

Glycoconj J (2011) 28:519–524
521
Scheme 1 Reagents: a N-Iodo-
succinimide (NIS), HClO4-SiO2,
CH2Cl2, MS 4Å, −30°C, 1 h,
87% for 6 and 77% for 9; b
CH3ONa, CH3OH, room tem-
perature, 2 h, quantitative; c
HClO4-SiO2, CH2Cl2, −10°C,
1 h, 78%; (d) H2, 20% Pd(OH)2-
C, CH3OH, room temperature,
24 h; e acetic anhydride, HClO4-
SiO2, room temperature, 1 h; f
CH3ONa, CH3OH, room tem-
perature, 6 h, 63% in three steps;
g acetic anhydride, HClO4-SiO2,
room temperature, 3 h; h TFA,
CH2Cl2, room temperature, 8 h,
58% in two steps
OR
O
Ph
O
AcO
O
OAc
B
BnO
O
D
AcO
OSE
O
Ph
O
OAc
O
BnO
AcO
a
O
N3
O
2 + 3
4 + 5
A
O
BnO
C
6: R = Ac
7
d, e, f
a
b
1
O
: R = H
Ph
O
O
O
AcO
OAc
B
g, h
10
BnO
c
O
D
AcO
O
Ph
O
O
BnO
OAc
O
BnO
AcO
O
N3
A
OSE
O
C
9
8
SEt
anhydride and HClO4-SiO2 and (b) treatment of the per-O-
acetylated tetrasaccharide derivative with trifluoroacetic
acid (TFA) to furnish per-O-acetylated tetrasaccharide
hemiacetal derivative (10) in 58% yield, which was
confirmed from its spectral analysis.
(100 mL). The organic layer was successively washed with
5% aq. Na2S2O3, NaHCO3 and water, dried (Na2SO4) and
concentrated under reduced pressure. The crude product
was purified over SiO2 using hexane-EtOAc (5:1) as eluant
to give pure compound 6 (1.9 g, 87%). White solid; m.p.
25
65°C; [α]D −15.3 (c 1.1, CHCl3); IR (KBr): 3465, 3091,
3065, 3034, 2953, 2927, 2871, 1751, 1606, 1497, 1455,
Experimental
1373, 1312, 1284, 1233, 1142, 1096, 1047, 970, 860,
1
697 cm−1; H NMR (500 MHz, CDCl3): δ 7.54–7.25 (m,
General methods All reactions were monitored by thin
layer chromatography over silica gel-coated TLC plates.
The spots on TLC were visualized by warming ceric
sulfate (2% Ce(SO4)2 in 1 M H2SO4)-sprayed plates on a
hot plate. Silica gel 230–400 mesh was used for column
20 H, Ar-H), 5.71–5.70 (m, 1 H, H-2B), 5.68 (s, 1 H,
PhCH), 5.61 (s, 1 H, PhCH), 5.27 (br s, 1 H, H-1B), 4.92
(d, J=12.4 Hz, 1 H, PhCH2), 4.76–4.67 (m, 3 H, PhCH2),
4.64–4.56 (m, 1 H, H-5B), 4.53 (br s, 1 H, H-1A), 4.37–4.33
(dd, J=10.4, 4.8 Hz, 1 H, H-6aA), 4.27–4.24 (dd, J=10.4,
4.6 Hz, 1 H, H-6aB), 4.22–4.17 (m, 2 H, H-2A, H-3B), 4.15
(t, J=9.6 Hz each, 1 H, H-4A), 4.04 (t, J=9.6 Hz each, 1 H,
H-4B), 4.00–3.95 (m, 1 H, OCH2), 3.93 (t, J=10.0 Hz each
1 H, H-6bA), 3.79 (t, J=10.0 Hz each, 1 H, H-6bB), 3.67
(dd, J=10.0, 2.8 Hz, 1 H, H-3A), 3.60–3.52 (m, 1 H,
OCH2), 3.40–3.33 (m, 1 H, H-5A), 2.14 (s, 3 H, COCH3),
1.20–1.05 (m, 2 H, CH2), 0.01 (br s, 9 H, Si(CH3)3); 13C
NMR (125 MHz, CDCl3): δ 169.9 (COCH3), 138.4–126.1
(Ar-C), 101.6 (PhCH), 101.5 (PhCH), 100.2 (C-1A), 99.9
(C-1B), 79.2 (C-3B), 78.5 (C-4B), 77.9 (C-3A), 74.5 (C-4A),
74.4 (C-2A), 73.1 (PhCH2), 72.4 (PhCH2), 69.5 (C-2B),
68.7 (C-6A), 68.6 (C-6B), 67.7 (C-5A), 67.3 (OCH2), 63.4
(C-5B), 21.1 (COCH3), 18.3 (CH2), −1.4 (Si(CH3)3); ESI-
MS: 863.3 [M+Na]+; Anal. Calcd. for C47H56O12Si
(840.35): C, 67.12; H, 6.71%; found: C, 66.93; H, 6.95%.
1
chromatography. H and 13C NMR, DEPT 135, 2D NMR
spectra were recorded on Brucker Avance DRX 400, 500
and 600 MHz spectrometers using CDCl3 and CD3OD as
solvents and TMS as internal reference unless stated
otherwise. Chemical shift values are expressed in δ ppm.
ESI-MS were recorded on a Micromass Quttro mass
spectrometer. Elementary analysis was carried out on
Carlo Erba-1108 analyzer. Optical rotations were mea-
sured at 25°C on a Jasco P-2000 polarimeter. Commer-
cially available grades of organic solvents of adequate
purity are used in all reactions. Silica supported perchloric
acid (HClO4-SiO2) was prepared following the earlier
report [14].
2-Trimethylsilylethyl (2-O-acetyl-3-O-benzyl-4,6-O-benzyli-
dene-α-D-mannopyranosyl)-(1→2)-3-O-benzyl-4,6-O-ben-
zylidene-β-D-mannopyranoside (6) To a solution of
compound 2 (1.2 g, 2.61 mmol) and compound 3 (1.3 g,
2.92 mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4
Å (2.0 g) and reaction mixture was cooled to −30°C. To the
cooled reaction mixture were added NIS (700.0 mg,
3.11 mmol) and HClO4-SiO2 (25.0 mg) and it was allowed
to stir for 1 h at the same temperature. The reaction mixture
was filtered through a Celite® bed and washed with CH2Cl2
2-Trimethylsilylethyl (3-O-benzyl-4,6-O-benzylidene-α-D-
mannopyranosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-β-
D-mannopyranoside (7) A solution of compound 6 (1.8 g,
2.14 mmol) in 0.1 M CH3ONa in CH3OH (25 mL) was
allowed to stir at room temperature for 2 h and neutralized
with Dowex 50 W X8 (H+) resin. The reaction mixture was
filtered, concentrated and passed through a short pad of
SiO2 to give pure compound 7 (1.7 g, quantitative). Yellow

522
Glycoconj J (2011) 28:519–524
(SCH2CH3); ESI-MS: 734.2 [M+Na]+; Anal. Calcd. for
C31H41N3O14S (711.23): C, 52.31; H, 5.81%; found: C,
52.10; H, 6.05%.
25
oil; [α]D −4.3 (c 1.0, CHCl3); IR (neat): 3470, 3089,
3064, 3033, 2953, 2928, 2336, 2109, 1955, 1887, 1727,
1
1497, 1378, 1215, 1098, 917, 837, 697 cm−1; H NMR
(500 MHz, CDCl3): δ 7.56–7.29 (m, 20 H, Ar-H), 5.66 (s,
1 H, PhCH), 5.62 (s, 1 H, PhCH), 5.41 (br s, 1 H, H-1B),
4.91–4.73 (m, 4 H, PhCH2), 4.62–4.55 (m, 1 H, H-5B),
4.54 (br s, 1 H, H-1A), 4.38 (dd, J=10.4, 4.0 Hz, 1 H, H-
6aA), 4.30 (br s, 1 H, H-2B), 4.29–4.25 (m, 1 H, H-6aB),
4.24 (br s, 1 H, H-2A), 4.15–4.08 (m, 3 H, H-3B, H-4A, H-
4B), 4.08–3.98 (m, 1 H, OCH2), 3.92 (t, J=10.0 Hz each,
1 H, H-6bA), 3.87 (t, J=10.0 Hz each, 1 H, H-6bB), 3.70
(dd, J=10.0, 2.8 Hz, 1 H, H-3A), 3.60–3.52 (m, 1 H,
OCH2), 3.40–3.32 (m, 1 H, H-5A), 2.77 (br s, 1 H, OH),
1.26–1.07 (m, 2 H, CH2), 0.04 (br s, 9 H, Si(CH3)3); 13C
NMR (125 MHz, CDCl3): δ 138.4–126.1 (Ar-C), 101.6
(PhCH), 101.5 (PhCH), 100.9 (C-1B), 100.4 (C-1A), 79.1
(C-4B), 79.0 (C-3B), 78.0 (C-3A), 75.9 (C-4A), 74.1 (C-2A),
73.1 (PhCH2), 73.0 (PhCH2), 69.9 (C-2B), 68.8 (C-6A),
68.7 (C-6B), 67.7 (OCH2), 67.3 (C-5A), 62.9 (C-5B), 18.3
(CH2), −1.4 (Si(CH3)3); ESI-MS: 821.3 [M+Na]+; Anal.
Calcd. for C45H54O11Si (798.34): C, 67.65; H, 6.81%;
found: C, 67.42; H, 7.00%.
2-Trimethylsilylethyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-
D-galactopyranosyl)-(1→4)-(2,3-di-O-acetyl-6-O-benzyl-
α-D-mannopyranosyl)-(1→2)-(3-O-benzyl-4,6-O-benzyli-
dene-α-D-mannopyranosyl)-(1→2)-3-O-benzyl-4,6-O-ben-
zylidene-β-D-mannopyranoside (9) To a solution of
compound 7 (1.0 g, 1.25 mmol) and compound
8 (980 mg, 1.37 mmol) in anhydrous CH2Cl2 (5 mL) was
added MS 4Å (1.0 g) and reaction mixture was cooled to
−30°C. To the cooled reaction mixture were added NIS
(350.0 mg, 1.55 mmol) and HClO4-SiO2 (15.0 mg) and it
was allowed to stir for 1 h at the same temperature. The
reaction mixture was filtered through a Celite® bed and
washed with CH2Cl2 (100 mL). The organic layer was
successively washed with 5% aq. Na2S2O3, NaHCO3 and
water, dried (Na2SO4) and concentrated under reduced
pressure. The crude product was purified over SiO2 using
hexane-EtOAc (4:1) as eluant to give pure compound 9
(1.4 g, 77%). White solid; m.p. 83°C; [α]D25=+ 42.3 (c 1.2,
CHCl3); IR (KBr): 3482, 3034, 2954, 2929, 1753, 1498,
1
Ethyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyra-
nosyl)-(1→4)-2,3-di-O-acetyl-6-O-benzyl-1-thio-α-D-man-
nopyranoside (8) A solution of compound 4 (1.3 g,
2.72 mmol) and compound 5 (1.0 g, 2.51 mmol) in
anhydrous CH2Cl2 (10 mL) was cooled to −10°C under
argon. To the cooled reaction mixture was added HClO4-
SiO2 (50.0 mg) and the reaction mixture was allowed to stir
at same temperature for 1 h. The reaction mixture was
filtered through a Celite® bed and concentrated under
reduced pressure. The crude product was purified over SiO2
using hexane-EtOAc (4:1) as eluant to furnish pure
compound 8 (1.4 g, 78%). Yellow oil; [α]D25+17.8 (c
1.2, CHCl3); IR (neat): 3481, 3031, 2873, 2113, 1752,
1498, 1373, 1237, 1130, 1045, 970, 850, 746, 699 cm−1; 1H
NMR (500 MHz, CDCl3): δ 7.37–7.24 (m, 5 H, Ar-H),
5.32–5.31 (m, 2 H, H-2C, H-3C), 5.30 (br s, 1 H, H-4D),
5.28 (br s, 1 H, H-4D), 5.24 (br s, 1 H, H-1C), 5.19 (dd, J=
10.6, 3.4 Hz, 1 H, H-3D), 4.65–4.59 (2 d, J=12.1 Hz, 2 H,
PhCH2), 4.32–4.29 (m, 1 H, H-5C), 4.11 (t, J=9.6 Hz, 1 H,
H-4C), 4.04–4.01 (m, 1 H, H-5D), 3.90–3.85 (m, 2 H, H-
1371, 1241, 1177, 1093, 972, 861, 698 cm−1; H NMR
(500 MHz, CDCl3): δ 7.49–7.19 (m, 25 H, Ar-H), 5.66 (s,
1 H, PhCH), 5.57 (s, 1 H, PHCH), 5.48–5.46 (m, 2 H, H-
2C, H-3C), 5.33 (br s, 1 H, H-1B), 5.32 (br s, 1 H, H-4D),
5.31 (d, J=2.4 Hz, 1 H, H-1D), 5.20 (dd, J=10.6, 3.0 Hz,
1 H, H-3D), 5.10 (br s, 1 H, H-1C), 4.90 (d, J=12.0 Hz, 1 H,
PhCH2), 4.76–4.70 (m, 2 H, PHCH2), 4.60 (d, J=12.4 Hz,
1 H, PhCH2), 4.52 (d, J=12.4 Hz, 1 H, PhCH2), 4.51–4.48
(m, 1 H, H-5B), 4.45 (d, J=12.0 Hz, 1 H, PhCH2), 4.43 (br
s, 1 H, H-1A), 4.33–4.29 (m, 1 H, H-6aA), 4.24 (br s, 1 H,
H-2B), 4.23–4.20 (m, 1 H, H-6aB), 4.18 (t, J=9.6 Hz each,
1 H, H-4B), 4.14 (t, J=9.7 Hz each, 1 H, H-4C), 4.12–4.10
(m, 1 H, H-3B), 4.09 (br s, 1 H, H-2A), 3.99 (t, J=9.6 Hz
each, 1 H, H-4A), 3.96–3.93 (m, 3 H, H-5C, H-5D, OCH2),
3.91–3.84 (m, 3 H, H-6aC, H-6bA, H-6bB), 3.80 (dd, J=11.5,
5.0 Hz, 1 H, H-6bC), 3.69 (dd, J=10.8, 3.4 Hz, 1 H, H-2D),
3.56 (dd, J=10.0, 2.8 Hz, 1 H, H-3A), 3.54–3.49 (m, 3 H,
H-6abD, OCH2), 3.31–3.28 (m, 1 H, H-5A), 2.10, 2.05, 2.04,
2.01, 1.94 (5 s, 15 H, 5 COCH3), 1.19–1.02 (m, 2 H, CH2),
0.00 (br s, 9 H, Si(CH3)3); 13C NMR (125 MHz, CDCl3): δ
170.1, 170.0, 169.9, 169.6, 169.5 (5 COCH3), 139.0–126.0
(Ar-C), 101.4 (PhCH), 101.3 (PhCH), 100.5 (C-1D), 100.2
(C-1A), 99.2 (C-1C), 98.5 (C-1B), 79.3 (C-4B), 78.9 (C-4A),
78.1 (C-3A), 77.1 (C-2B), 75.8 (C-3B), 74.4 (C-2A), 73.2
(PhCH2), 72.9 (PhCH2), 72.7 (PhCH2), 72.1 (C-3C), 71.9
(C-4C), 70.7 (C-5D), 69.6 (C-2C), 68.8 (C-6A), 68.7 (C-6B),
68.6 (C-6D), 68.0 (C-3D), 67.6 (OCH2), 67.2 (C-5A), 67.1
(C-4D), 66.9 (C-5C), 63.6 (C-5B), 61.2 (C-6C), 57.4 (C-2D),
20.8, 20.7, 20.6, 20.5 (2 C) (5 COCH3), 18.2 (CH2), −1.50
(Si(CH3)3); ESI-MS: 1470.5 [M+Na]+; Anal. Calcd. for
6
abC), 3.83 (dd, J=11.5, 5.2 Hz, 1 H, H-6aD), 3.72 (dd, J=
11.5, 1.8 Hz, 1 H, H-6bD), 3.66 (dd, J=10.7, 3.7 Hz, 1 H,
H-2D), 2.68–2.58 (m, 2 H, SCH2CH3), 2.09, 2.01, 1.98,
1.97 (4 s, 15 H, 5 COCH3), 1.25 (t, J=7.4 Hz each, 3 H,
SCH2CH3); 13C NMR (125 MHz, CDCl3): δ 170.1, 170.0,
169.9, 169.7, 169.5, 5 COCH3, 138.3–127.6 (Ar-C), 99.0
(C-1D), 81.9 (C-1C), 73.2 (PhCH2), 73.0 (C-4C), 72.6 (C-
4D), 71.6 (C-2C), 70.8 (C-5C), 69.2 (C-6D), 68.1 (C-3D),
67.5 (C-3C), 67.4 (C-5D), 61.6 (C-6C), 57.6 (C-2D), 25.6
(SCH2CH3), 21.1, 21.0, 20.9, 20.8, 20.7 (5 COCH3), 14.8

Glycoconj J (2011) 28:519–524
523
C74H89N3O25Si (1447.56): C, 61.36; H, 6.19%; found: C,
61.15; H, 6.43%.
removed and co-evaporated with toluene (3×10 mL) under
reduced pressure. To a solution of the acetylated product in
CH2Cl2 (5 mL) was added trifluoroacetic acid (0.3 mL) and
the reaction mixture was allowed to stir at room tempera-
ture for 8 h. The solvents were removed and co-evaporated
with toluene (3×10 mL) under reduced pressure. The crude
product was passed through a short pad of SiO2 to give
pure compound 10 (85 mg, 58%) as a mixture of α- and β-
isomers (2:1). 1H NMR (CDCl3, 400 MHz): α/β-isomers: δ
5.43–5.40 (m, 3 H), 5.39–5.29 (m, 6 H), 5.27–5.16 (m,
3 H), 5.14–5.05 (m, 3 H), 4.94–4.92 (m, 1.5 H), 4.67–4.62
(m, 1.5 H), 4.51–4.45 (m, 2 H), 4.31–3.95 (m, 22 H), 2.14,
2.11, 2.10, 2.09, 2.07, 2.06, 2.04 (7 s, 39 H, 13 COCH3α),
2.02, 2.01, 2.00, 1.99, 1.97, 1.96, 1.94 (7 s, 19.5 H, 13
COCH3β); 13C NMR (CDCl3, 100 MHz): α/β-isomer: δ
171.1–169.9 (CH3CO), 99.3 (C-1Dα), 99.1 (C-1Dβ), 98.9
(C-1Bβ), 98.8 (C-1Cβ), 98.7 (2 C, C-1Bα, C-1Cα), 98.3 (C-
2-Trimethylsilylethyl (2-acetamido-2-deoxy-α-D-galacto-
pyranosyl)-(1→4)-(α-D-mannopyranosyl)-(1→2)-(α-D-
mannopyranosyl)-(1→2)-β-D-mannopyranoside (1) To a
solution of compound 9 (1.0 g, 0.69 mmol) in CH3OH-
EtOAc (20 mL; 10:1 v/v) was added 20% Pd(OH)2-C
(200.0 mg) and the reaction mixture was allowed to stir at
room temperature under a positive pressure of hydrogen for
24 h. The reaction mixture was filtered through a Celite®
bed and evaporated to dryness. To a solution of the crude
product in acetic anhydride (3 mL) was added HClO4-SiO2
(50.0 mg) and the reaction mixture was stirred at room
temperature for 1 h. The reaction mixture was filtered, and
solvents were removed under reduced pressure. A solution
of the acetylated product in 0.1 M CH3ONa in CH3OH
(25 mL) was allowed to stir at room temperature for 6 h and
neutralized with Dowex 50 W X8 (H+) resin. The reaction
mixture was filtered, concentrated and passed through a
column of Sephadex® LH-20 using CH3OH-H2O (80 mL;
4:1 v/v) as eluant to give pure compound 1 (350.0 mg,
63%). White powder; [α]D25+30.4 (c 1.0, CH3OH); IR
(KBr): 3392, 2940, 2510, 2106, 1643, 1379, 1068, 1027,
792, 697 cm−1; 1H NMR (500 MHz, CD3OD): δ 5.32 (br s,
1 H, H-1B), 5.17 (d, J=3.6 Hz, 1 H, H-1D), 5.00 (br s, 1 H,
H-1C), 4.52 (br s, 1 H, H-1A), 4.25 (dd, J=10.8, 3.6 Hz,
1 H, H-2D), 4.07–4.04 (m, 1 H, H-5C), 4.02–4.01 (m, 1 H,
H-2B), 3.97–3.96 (m, 1 H, H-2A), 3.95–3.92 (m, 3 H, H-2C,
H-5B, OCH2), 3.91–3.89 (m, 2 H, H-3B, H-4C), 3.87–3.85
(m, 2 H, H-3D, H-6aA), 3.84–3.79 (m, 2 H, H-4A, H-6ac),
3.78–3.68 (m, 6 H, H-4B, H-5D, H-6abD, H-6bA), 3.67–3.65
(m, 3 H, H-6bC, H-6abB), 3.56–3.50 (m, 3 H, H-3A, H-3C,
OCH2), 3.22–3.18 (m, 1 H, H-5A), 1.99 (s, 3 H, COCH3),
0.96–0.94 (m, 2 H, CH2), 0.00 (m, 9 H, (Si(CH3)3); 13C
NMR (125 MHz, CD3OD): δ 174.8 (COCH3), 103.5 (C-
1C), 101.5 (C-1B), 101.1 (2 C, C-1A, C-1D), 79.2 (C-2B),
78.8 (C-5A), 77.4 (C-2A), 77.3 (C-4A), 76.1 (C-3C), 73.8
(C-5C), 73.7 (C-5D), 73.4 (C-3B), 72.6 (C-5B), 72.3 (2 C, C-
2C, C-4C), 70.8 (C-4D), 70.5 (C-3D), 69.1 (C-3A), 68.6 (C-
4B), 68.1 (OCH2), 63.1 (C-6A), 63.0 (C-6D), 62.7 (C-6B),
62.5 (C-6C), 52.3 (C-2D), 23.0 (COCH3), 19.3 (CH2); ESI-
MS: 830.3 [M+Na]+; Anal. Calcd. for C31H57NO21Si
(807.32): C, 46.09; H, 7.11%; found: C, 46.30; H, 7.37%.
1
Aβ), 93.1 (C-1Aα), 77.4 (2 C, α), 76.2 (2 C, β), 76.1 (2 C,
β), 73.4 (β), 72.3 (β), 71.7 (2 C, β), 71.6 (α), 71.5 (2 C,
α,β), 70.4 (β), 70.3 (α), 70.2 (α), 69.6 (2 C, β), 69.5 (2 C,
α), 69.3 (α), 68.7 (α), 68.3 (β), 67.9 (2 C, α), 67.8 (α),
67.7 (3 C, β), 67.5 (2 C, α), 67.4 (β), 66.9 (β), 66.8 (α),
63.8 (α), 63.5 (β), 62.8 (α), 62.7 (α), 62.5 (β), 62.0 (α),
20.9–20.7 (COCH3).
Acknowledgements G. G. thanks CSIR, New Delhi for providing a
Senior Research fellowship. This project was supported by Bose
Institute, Kolkata.
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