Convergent synthesis of the pentasaccharide repeating unit of the O-antigenic polysaccharide of enterohaemorrhagic Escherichia coli O113

Article abstract of DOI:10.1007/s10719-012-9383-4

An acidic pentasaccharide repeating unit corresponding to the O-antigenic polysaccharide of enterohaemorrhagic Escherichia coli O113 as its p-methoxyphenyl glycoside has been synthesized in a convergent manner by adopting a [3+2] block glycosylation strategy. During the synthetic endeavor a one-pot reaction condition for stereoselective glycosylation and protecting group manipulation has been applied. All glycosylation steps are highly stereoselective with good to excellent yield.

Full text of DOI:10.1007/s10719-012-9383-4

Glycoconj J (2012) 29:181–188
DOI 10.1007/s10719-012-9383-4
Convergent synthesis of the pentasaccharide repeating unit
of the O-antigenic polysaccharide of enterohaemorrhagic
Escherichia coli O113
Abhishek Santra & Anup Kumar Misra
Received: 6 March 2012 /Revised: 16 April 2012 /Accepted: 18 April 2012 /Published online: 5 May 2012
#
Springer Science+Business Media, LLC 2012
Abstract An acidic pentasaccharide repeating unit
corresponding to the O-antigenic polysaccharide of enter-
ohaemorrhagic Escherichia coli O113 as its p-methoxy-
phenyl glycoside has been synthesized in a convergent
manner by adopting a [3+2] block glycosylation strategy.
During the synthetic endeavor a one-pot reaction condition
for stereoselective glycosylation and protecting group
manipulation has been applied. All glycosylation steps are
highly stereoselective with good to excellent yield.
haemorrhagic uremic syndrome. The most cited EHEC
strain is O157:H7, which is associated with several diar-
rhoeal outbreaks in the developed countries [5, 6]. Besides
this, several EHEC strains have been identified for their
pathogenic potential to cause severe diarrhoral infections,
which include E. coli O4, O5, O16, O26, O46, O48, O55,
O91, O98, O111ab, O113, O117, O118, O119, O125, O126,
O128, O145 etc. [7]. It has been established that the bacte-
rial virulence comes up from the O-specific polysaccharides
(O-antigens) present in their cell membrane. As a conse-
quence, a large number of reports appeared on the structural
characterization of the O-antigens of various pathogenic
bacteria as well as their application in the development of
glycoconjugate based therapeutics [8–10]. The structure of
the pentasaccharide repeating unit of the O-specific lipo-
polysaccharide of E. coli O113 has been established by
Parolis et al. (Fig. 1) [11]. It is an acidic oligosaccharide
having a D-galacturonic acid moiety with three 1,2-cis gly-
cosyl linkages present in it.
Cell wall O-antigens are considered as important class of
molecules for the development of glycoconjugate based
therapeutics. It would be pertinent to carry out several
biological experiments using a pure pentasaccharide frag-
ment to establish its pathological implications in the diar-
rhoeal infections. Development of a chemical synthetic
strategy for the preparation of pure pentasaccharide would
always be welcome to avoid the troublesome isolation and
purification of the pentasaccharide from the natural source.
In this context, most of the currently available vaccines
contain attenuated live strains of bacteria or isolated poly-
saccharides, there could be a chance of self infection or
contaminations respectively. Replacement of the live strains
or natural polysaccharides with synthetic oligosaccharides
or glycoconjugates could reduce the abovementioned short-
comings as well as could improve the access to the vaccines
.
.
Keywords Enterohaemorrhagic Escherichia coli O113
.
.
Pentasaccharide O-antigen Glycosylation
Introduction
Enterohaemorrhagic Escherichia coli (EHEC) are one of the
major causative agents of diarrhoea with life-threatening
complications e.g. haemorrhagic colitis and haemorrhagic
uremic syndrome [1, 2]. EHEC acquire their virulent action
due to the release of vero-toxin or Shiga-toxin and hence are
also called Verotoxigenic E. coli (VTEC) [3]. The action of
the Shiga-toxin released by the EHEC on the endothelial
cells of the host leads to the pathological lesions associated
with haemorrhagic colitis and haemorrhagic uremic syn-
drome [4]. The young and elderly people are susceptible
for the EHEC infections and suffer from diarrhoea, and
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-012-9383-4) contains supplementary material,
which is available to authorized users.
:
A. Santra A. K. Misra (*)
Division of Molecular Medicine, Bose Institute,
P-1/12, C.I.T. Scheme VII M,
Kolkata 700054, India
e-mail: akmisra69@gmail.com

182
Glycoconj J (2012) 29:181–188
its removal by tuning the glycosylation reaction condition in
one-pot increases overall yield by reducing the number of
steps [17]; (b) stereoselective [3+2] block glycosylation
reduces the number of steps and improves overall yield;
(c) application of perchloric acid supported over silica
(HClO4-SiO2) as a heterogeneous acid catalyst in the gly-
cosylation of trichloroacetimidate derivative and thioglyco-
side; benzylidene acetal formation and direct conversion of
benzylidene acetal to O-acetylated derivative allowed to
avoid the use of corrosive, moisture sensitive triflic acid or
other protic acids and harsh reaction conditions; (d) the D-
galactosamine moiety has been derived from tri-O-acetyl-D-
galactal using azido-nitration technique reported earlier
[18]; (e) preparation of the α-D-galacturonic acid moiety
at the late stage of the synthetic strategy using a TEMPO
mediated selective oxidation protocol under phase transfer
reaction condition.
Fig. 1 Structure of the repeating unit of the O-antigenic lipopolysac-
charise of E. coli O113
with higher potency. As a part of our ongoing program for
the synthesis of complex oligosaccharides from microbial
origin, an efficient chemical synthesis of the pentasacchar-
ide corresponding to the O-antigen of the enterohaemorrha-
gic E. coli O113 is presented (Fig. 2). p-Methoxyphenyl
group could act as a temporary anomeric protecting group
and can be removed as and when necessary.
Results and discussion
p-Methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-β-D-glucopyranoside (2) was allowed to cou-
ple with ethyl 2-O-benzyl-4,6-O-benzylidene-3-O-(4-
methoxybenzyl)-1-thio-β-D-galactopyranoside (3) in the
presence of a combination of N-iodosuccinimide (NIS) and
trifluoromethane sulfonic acid (TfOH) [17, 19, 20]. Con-
trolling the reaction condition initially at low temperature
and then at high temperature furnished the disaccharide
derivative (7) in a 72 % yield, in which stereoselective
glycosylation and removal of p-methoxybenzyl group was
achieved in one pot. A minor quantity (∼8 %) of 1,2-trans
isomer of compound 7 was also formed, which was sepa-
rated by column chromatography. It is worth mentioning
that after stereoselective glycosylation at low temperature,
the elevation of temperature resulted in the clean removal of
p-methoxybenzyl group by TfOH present in the reaction
medium. Appearance of signals in the NMR spectra con-
firmed the formation of compound 7 [signals at δ 5.70 (d,
J08.5 Hz, H-1A), 5.48 (d, J03.5 Hz, H-1B) in 1H NMR and
δ 98.1 (C-1B), 97.8 (C-1A) in the 13C NMR spectra]
(Scheme 1).
The synthesis of the pentasaccharide 1 as its p-methoxy-
phenyl glycoside was carried out using a convergent [3+2]
block glycosylation strategy. A disaccharide glycosyl accep-
tor (7) was stereoselectively condensed with a trisaccharide
glycosyl donor (12). The disaccharide derivative (7) and the
trisaccharide derivative (12) were prepared from suitably
protected monosaccharide intermediates 2 [12], 3 [13], 4
[14], 5 [15] and 6 [16] prepared from the commercially
available reducing sugars using reported literature (Fig. 2).
The presence of α-D-galactosamine, α-D-galacturonic acid
and α-D-galactose moieties make the synthesis of the target
molecule challenging. A number of noteworthy features in
the present synthetic strategy, such as: (a) application of p-
methoxybenzyl ether as a temporary protecting group and
OH
OH
HO
HO
O
O
O
E
D
HO
OH
AcHN
O
O
ONa
O
HO
OH
C
HO
O
HO
B
In another set of experiments, stereoselective glycosyla-
tion of p-methoxyphenyl 2,3,6-tri-O-benzyl-β-D-galacto-
pyranoside (4) with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-
D-galactopyranosyl trichloroacetimidate (5) in the presence
O
NHAc
O
OPMP
HO
O
A
HO
1
PMP: p-Methoxyphenyl
OH
Ph
Ph
O
O
OBn
O
HO
O
O
O
A
Ph
OPMP
NPhth
HO
O
C
BnO
B
OPMP
MBnO
SEt
O
O
OBn
2
OBn
4
3
O
a
B
OAc
AcO
HO
2 + 3
NPhth
O
OAc
AcO
O
OPMP
BnO
D
AcO
O
A
O
NH
E
AcO
SEt
O
N3
O
CCl3
O
OAc
Ph
5
6
7
Fig. 2 Structure of the synthesized pentasaccharide (1) and its syn-
thetic precursors
Scheme 1 Reagents: (a) NIS, TfOH, CH2Cl2, MS 4 Å, −30 °C for
45 min, then 0 °C for 30 min, 72 %

Glycoconj J (2012) 29:181–188
183
of perchloric acid supported over silica (HClO4-SiO2) [21]
in a mixture of dichloromethane-diethyl ether furnished the
disaccharide derivative (8) in a 64 % yield. The moderate
yield of the reaction may be explained considering the less
reactivity of the 4-hydroxyl group of the D-galactose deriv-
ative (4). The formation of compound 8 was supported by
spectral analysis [signals at δ 4.95 (d, J03.5 Hz, H-1D), 4.72
Spectral analysis of compound 13 confirmed its formation
[signals at δ 6.76 (d, J08.5 Hz, H-1A), 5.48 (d, J03.5 Hz,
H-1B), 5.26 (d, J03.0 Hz, H-1C), 5.08 (d, J03.5 Hz, H-1D),
4.52 (d, J08.0 Hz, H-1E) in the 1H NMR and δ 100.7 (JC-1/
H-10158.0 Hz) (C-1E), 98.3 (JC-1/H-10170.0 Hz) (C-1B),
98.1 (JC-1/H-10160.0 Hz) (C-1A), 98.0 (JC-1/H-10169.0 Hz)
(C-1D), 91.7 (JC-1/H-10172.0 Hz) (C-1C) in the 13C NMR
spectra]. Appearance of JC-1/H-1 values 158.8 and 160.0 Hz
indicated the presence of two equatorial or β-glycosyl link-
ages and JC-1/H-1 values 169.0, 170.0 and 172.0 Hz indicated
the presence of three axial or α-glycosyl linkages in the
compound 13 [27, 28]. Compound 13 was subjected to a
series of reactions involving (a) reduction of azido group
and selective removal of benzyl groups by controlled hy-
drogenation [12, 29] over Pd(OH)2-C followed by N-acety-
lation; (b) selective TEMPO mediated oxidation of the
primary hydroxyl group to the carboxylic group under a
phase transfer reaction condition [30, 31]; (c) removal of
N-phthalimido group by hydrazinolysis [32] followed by
acetylation using acetic anhydride and pyridine; (d) removal
of benzylidene acetal by hydrogenation and finally (e) de-O-
acetylation using sodium methoxide to furnish target penta-
saccharide 1, which was purified over Sephadex® LH-20
column using (CH3OH-H2O; 4:1) as eluant to give pure
compound 1 in a 51 % yield. Spectral analysis of compound
1 unambiguously confirmed its formation [signals at δ 5.15
(br s, H-1C), 4.90 (br s, H-1B, H-1D), 4.80–4.78 (m, H-1A),
1
(d, J07.5 Hz, H-1C) in the H NMR and δ 103.2 (C-1C),
98.5 (C-1D) in the 13C NMR spectra]. Saponification of
compound 8 using sodium methoxide followed by 4,6-O-
benzylidene acetal formation using benzaldehyde dimethyl
acetal in the presence of HClO4-SiO2 [22] furnished disac-
charide derivative 9 in a 77 % yield. Stereoselective glyco-
sylation of compound 9 with ethyl 2,3,4,6-tetra-O-acetyl-1-
thio-β-D-galactopyranoside (6) in the presence of a combi-
nation of NIS and HClO4-SiO2 in dichloromethane [23]
afforded trisaccharide derivative 10 in a 78 % yield. Appear-
ance of signals in the NMR spectra confirmed the formation of
compound 10 [signals at δ 5.01 (d, J03.5 Hz, H-1D), 4.78 (d,
1
J08.0 Hz, H-1E), 4.76 (d, J07.5 Hz, H-1C) in the H NMR
and δ 103.4 (C-1E), 103.4 (C-1C), 99.6 (C-1D) in the 13C
NMR spectra]. In a one-pot de-benzylidenation and O-acety-
lation reaction condition [24], compound 10 was treated with
acetic anhydride in the presence HClO4-SiO2 to give com-
pound 11 in 80 % yield. Oxidative removal of p-methoxy-
phenyl group in compound 11 using ceric ammonium (IV)
nitrate (CAN) [25] followed by the reaction of the resulting
hemiacetal with trichloroacetonitrile in the presence of DBU
[26] led to the formation of a α/β mixture of trisaccharide
trichloroacetimidate derivate 12 in 74 % yield, which was
immediately used in the next step (Scheme 2).
1
4.32 (d, J08.0 Hz, H-1E) in the H NMR and δ 104.9 (C-
1E), 102.6 (C-1A), 99.1 (2 C, C-1B, C-1D), 96.1 (C-1C) in the
13C NMR spectra] (Scheme 3).
Stereoselective glycosylation of compound 7 with com-
pound 12 in the presence of HClO4-SiO2 [21] as a hetero-
geneous catalyst in a mixture of dichloromethane-diethyl
ether furnished pentasaccharide derivative 13 in a 68 %
yield together with minor quantity (∼10 %) of it’s another
isomer, which was separated by column chromatography.
Conclusion
In conclusion, a straight forward convergent synthetic strat-
egy has been developed for the synthesis of the pentasac-
charide repeating unit corresponding to the O-antigen of
Scheme 2 Reagents: (a)
HClO4-SiO2, CH2Cl2-Et2O,
−20 °C, 1 h, 64 %; (b) 0.1 M
CH3ONa, CH3OH, room
temperature, 2 h; (c)
benzaldehyde dimethylacetal,
HClO4-SiO2, CH3CN-DMF,
room temperature, 5 h, 77 % in
two steps; (d) NIS, HClO4-
SiO2, CH2Cl2, MS 4 Å, −40 °C,
45 min, 78 %; (e) acetic
anhydride, HClO4-SiO2, room
tempearature, 30 min, 80 %; (f)
CAN, CH3CN-H2O, room
temperature, 2 h; (g) CCl3CN,
CH2Cl2, DBU, −20 °C, 1 h,
74 % in two steps
Ph
O
OAc
AcO
O
O
O
D
D
AcO
HO
6
d
N
a
b,c
N
3 O
OBn
O
OBn
O
3 O
4 + 5
C
C
BnO
BnO
OPMP
OPMP
OBn
OBn
9
8
OAc
OR2
R1O
OAc
AcO
OAc
AcO
AcO
O
O
O
O
E
E
D
D
AcO
AcO
O
O
f,g
OAc
OAc
N
3 O
N
3 O
OBn
O
OBn
O
C
C
NH
CCl3
BnO
BnO
OPMP
BnO
BnO
O
10: R1; R2 = PhCH
e
12
: R1 = R2 = Ac
11

184
Glycoconj J (2012) 29:181–188
OAc
OAc
AcO
AcO
O
O
O
E
D
AcO
Ph
OAc
N
3 O
OBn
b,c,d,e,f,g,h
NPhth
a
1
7 + 12
O
O
O
C
BnO
O
BnO
B
O
OPMP
BnO
A
O
O
O
13
O
Ph
Scheme 3 Reagents: (a) HClO4-SiO2, CH2Cl2-Et2O, −10 °C, 1 h,
68 %; (b) H2, 20 % Pd(OH)2-C, CH3OH, room temperature, 4 h; (c)
acetic anhydride, CH3OH, room temperature, 1 h; (d) (i) TEMPO,
NaBr, TBAB, NaHCO3, NaOCl, CH2Cl2, H2O, 5 °C, 3 h; (ii) NaClO2,
tert-butanol, 2-methyl-2-butene, NaH2PO4, room temperature, 3 h; (e)
NH2NH2·H2O, EtOH, 80 °C, 7 h; (f) acetic anhydride, pyridine, room
temperature, 2 h; (g) H2, 20 % Pd(OH)2-C, CH3OH, room temperature,
24 h; (h) 0.1 M CH3ONa, CH3OH, room temperature, 3 h, 51 %
enterohaemorrhagic E. coli O113. A one-pot reaction for the
glycosylation and removal of p-methoxybenzyl ether has
been adopted. HClO4-SiO2 has been used as an effective
acid catalyst in a number of reaction steps such as benzyli-
dene acetal formation, direct conversion of benzylidene
acetal to O-acetylated derivative and to activate glycosyl
trichloroacetimidate derivative and thioglycoside in combi-
nation with NIS avoiding the use of moisture sensitive
protic acids. A [3+2] block glycosylation technique has
been used. A late stage selective TEMPO mediated oxida-
tion protocol has been applied for the preparation of D-
galacturonic acid moiety. Over all, the target compound 1
was efficiently synthesized in a minimum number of steps.
compound 2 (1.0 g, 1.98 mmol) and compound 3 (1.2 g,
2.29 mmol) in anhydrous CH2Cl2 (8 mL) was added MS
4 Å (1 g) and the reaction mixture was stirred at room
temperature under argon for 30 min. The reaction mixture
was cooled to −30 °C and N-iodosuccinimide (NIS;
570.0 mg, 2.53 mmol) and TfOH (20 μL) were added to
it. After stirring the reaction mixture at −30 °C for 45 min
the temperature was raised to 0 °C and the reaction mixture
was allowed to stir at 0 °C for 30 min. The reaction mixture
was filtered through a Celite® bed and washed with CH2Cl2
(150 mL). The organic layer was successively washed with
5 % Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and
concentrated. The crude product was purified over SiO2
using hexane-EtOAc (4:1) as eluant to give pure compound
7 (1.2 g, 72 %). Colourless oil; [α]D25 +92.6 (c 1.2, CHCl3);
IR (neat): 3033, 2980, 2933, 2876, 1780, 1742, 1717, 1502,
Experimental
1459, 1400, 1099, 1031, 996, 699, cm−1 1H NMR
;
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 2N H2SO4)-sprayed plates on a hot plate.
Silica gel 230–400 mesh was used for column chromatog-
raphy. 1H and 13C NMR, DEPT 135, 2D COSY, HMQC and
gated 1H coupled 13C NMR spectra were recorded on
Brucker Avance DRX 500 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 analyzer. Optical rotations were measured at 25 °
C on a Jasco P-2000 polarimeter. Commercially available
grades of organic solvents of adequate purity are used in all
reactions. Perchloric acid supported over silica (HClO4-
SiO2) was prepared following the protocol reported by
Chakraborty et al. [33].
(500 MHz, CDCl3): δ 7.82–6.89 (m, 19 H, Ar-H), 6.74 (d,
J09.0 Hz, 2 H, Ar-H), 6.64 (d, J09.0 Hz, 2 H, Ar-H), 5.70
(d, J08.5 Hz, 1 H, H-1A), 5.48 (d, J03.5 Hz, 1 H, H-1B),
5.25 (s, 1 H, PhCH), 5.14 (s, 1 H, PhCH), 4.87 (t, J09.5 Hz,
1 H each, H-3A), 4.54 (t, J09.0 Hz each, 1 H, H-2A), 4.38
(d, J012.5 Hz, 1 H, PhCH2), 4.28–4.25 (m, 1 H, H-5A),
4.10 (d, J012.5 Hz, 1 H, PhCH2), 3.86 (dd, J09.5, 3.0 Hz, 1
H, H-3B), 3.82 (t, J09.0 Hz each, 1 H, H-4A), 3.79 (d, J0
3.0 Hz, 1 H, H-4B), 3.76–3.70 (m, 2 H, H-6abA), 3.64 (s, 3
H, OCH3), 3.57 (dd, J010.0, 3.5 Hz, 1 H, H-2B), 3.36 (d,
J012.0 Hz, 1 H, H-6aB), 2.95 (br s, 1 H, H-5B), 2.93 (d, J0
12.0 Hz, 1 H, H-6bB); 13C NMR (125 MHz, CDCl3): δ
155.7–114.5 (Ar-C), 102.1 (PhCH), 100.9 (PhCH), 98.1
(C-1B), 97.8 (C-1A), 82.3 (C-4A), 75.5 (C-4B), 74.9 (C-
2B), 73.8 (C-3A), 71.2 (PhCH2), 68.7 (C-6B), 68.5 (C-5A),
67.4 (C-3B), 66.1(C-6A), 62.9 (C-5B), 55.6 (C-2A), 55.3
(OCH3); ESI-MS: 866.2 [M+Na]+; Anal. Calcd. for
C48H45NO13 (843.29): C, 68.32; H, 5.37; found: C, 68.10;
H, 5.60.
p-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-α-D-gal-
actopyranosyl)-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-β-D-glucopyranoside (7) To a solution of
p-Methoxyphenyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-
galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-

Glycoconj J (2012) 29:181–188
185
galactopyranoside (8) A solution of compound 4 (1.4 g,
2.51 mmol) and compound 5 (1.9 g, 4.0 mmol) in anhydrous
CH2Cl2-Et2O (12 mL, 1:1 v/v) was cooled to −20 °C. To the
cooled reaction mixture was added HClO4-SiO2 (200 mg)
and the reaction mixture was allowed to stir at same tem-
perature for 1 h. The reaction mixture was filtered and
washed with CH2Cl2 (100 mL). The combined organic layer
was washed with satd. NaHCO3 and water, dried (Na2SO4)
and concentrated. The crude product was purified over SiO2
using hexane-EtOAc (5:1) as eluant to give pure compound
8 (1.4 g, 64 %). Colourless oil; [α]D25 +73.8 (c 1.2, CHCl3);
IR (neat): 3024, 2936, 2362, 2100, 1751, 1374, 1212, 1043,
11.5 Hz, 1 H, PhCH2), 4.82 (d, J011.5 Hz, 1 H, PhCH2),
4.78 (d, J08.0 Hz, 1 H, H-1C), 4.66–4.61 (2 d, J012.0 Hz
each, 2 H, PhCH2), 4.51–4.43 (2 d, J011.5 Hz each, 2 H,
PhCH2), 4.15 (d, J02.5 Hz, 1 H, H-4D), 5.30 (dd, J010.5,
3.0 Hz, 1 H, H-3D), 4.07 (br s, 1 H, H-5C), 4.05 (d, J0
3.0 Hz, 1 H, H-4C), 3.95–3.92 (m, 1 H, H-6aC), 3.78 (t, J0
10.0 Hz each, 1 H, H-2C), 3.69 (s, 3 H, OCH3), 3.60–3.51
(m, 4 H, H-2D, H-5D, H-6aD, H-6bC), 3.43 (dd, J010.0,
3.0 Hz, 1 H, H-3C), 3.33–3.30 (m, 1 H, H-6bD); 13C NMR
(125 MHz, CDCl3): δ 155.3–114.5 (Ar-C), 103.3 (C-1C),
101.0 (PhCH), 99.5 (C-1D), 80.6 (C-3C), 78.1 (C-2C), 75.6
(C-4C), 75.0 (PhCH2), 73.6 (PhCH2), 73.3 (C-4D), 73.1 (C-
5D), 72.7 (PhCH2), 68.9 (C-6C), 67.8 (C-3D), 67.0 (C-6D),
62.7 (C-5C), 61.4 (C-2D), 55.7 (OCH3); ESI-MS: 854.3 [M
+Na]+; Anal. Calcd. for C47H49N3O11 (831.34): C, 67.86;
H, 5.94; found: C, 67.65; H, 6.18.
1
767, 670 cm−1; H NMR (500 MHz, CDCl3): δ 7.30–7.15
(m, 15 H, Ar-H), 6.90 (d, J09.0 Hz, 2 H, Ar-H), 6.70 (d, J0
9.0 Hz, 2 H, Ar-H), 5.36 (d, J02.0 Hz, 1 H, H-4D), 5.30 (dd,
J010.5, 3.5 Hz, 1 H, H-3D), 4.95 (d, J03.5 Hz, 1 H, H-1D),
4.94 (d, J011.0 Hz, 1 H, PhCH2), 4.82 (d, J011.0 Hz, 1 H,
PhCH2), 4.72 (d, J07.5 Hz, 1 H, H-1C), 4.70–4.65 (m, 2 H,
H-5D), 4.45 (m, 1 H, PhCH2), 4.08 (d, J03.0 Hz, 1 H, H-
4C), 3.90–3.83 (m, 3 H, H-5C, H-6aC, H-6aD), 3.67 (s, 3 H,
OCH3), 3.60 (dd, J011.0, 4.0 Hz, 1 H, H-2D), 3.58–3.52 (m,
2 H, H-2C, H-6bD), 3.48–3.45 (m, 1 H, H-6bC), 3.40 (dd, J0
10.0, 3.0 Hz, 1 H, H-3C), 2.02, 1.97, 1.80 (3 s, 9 H, 3
COCH3); 13C NMR (125 MHz, CDCl3): δ 170.1 (2 C),
169.8 (3 COCH3), 155.7–114.5 (Ar-C), 103.2 (C-1C), 98.5
(C-1D), 79.7 (C-3C), 78.7 (C-2C), 75.2 (PhCH2), 73.8 (C-
4C), 73.5 (PhCH2), 73.3 (PhCH2), 72.9 (C-5C), 68.8 (C-3D),
67.3 (C-4D), 67.0 (C-6D), 66.2 (C-5D), 60.6 (C-6C), 58.0 (C-
2D), 55.6 (OCH3), 20.2, 20.6 (2 C) (3 COCH3); ESI-MS:
892.3 [M+Na]+; Anal. Calcd. for C46H51N3O14 (869.34): C,
63.51; H, 5.91; found: C, 63.28; H, 6.15.
p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyra-
nosyl)-(1→3)-(4,6-O-benzylidene-2-azido-2-deoxy-α-D-
galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-galacto-
pyranoside (10) To a solution of compound 9 (800.0 mg,
0.96 mmol) and compound 6 (450.0 mg, 1.14 mmol) in
anhydrous CH2Cl2 (5 mL) was added MS 4 Å (1.0 g) and
the reaction mixture was stirred at room temperature under
argon for 30 min. The reaction mixture was cooled to −40 °C
and NIS (280.0 mg, 1.24 mmol) and HClO4-SiO2 (15.0 mg)
were added to it and the reaction mixture was allowed to stir at
same temperature for 45 min. The reaction mixture was fil-
tered through a Celite® bed and washed with CH2Cl2
(50 mL). The organic layer was successively washed with
5 % Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and
concentrated. The crude product was purified over SiO2 using
hexane-EtOAc (5:1) as eluant to give pure compound 10
(870.0 mg, 78 %). Colourless oil; [α]D25 +80 (c 1.2, CHCl3);
IR (neat): 3474, 2932, 2868, 2117, 1757, 1502, 1226, 1100,
1079, 1056, 737, 699 cm−1; 1H NMR (500 MHz, CDCl3): δ
7.42–7.17 (m, 20 H, Ar-H), 6.94 (d, J09.0 Hz, 2 H, Ar-H),
6.72 (d, J09.0 Hz, 2 H, Ar-H), 5.33 (d, J03.5 Hz, 1 H, H-4E),
5.31 (s, 1 H, PhCH), 3.40 (dd, J08.0 Hz each, 1 H, H-2E),
5.01 (d, J03.5 Hz, 1 H, H-1D), 4.97 (d, J011.5 Hz, 1 H,
PhCH2), 4.95 (dd, J010.5, 3.0 Hz, 1 H, H-3E), 4.84 (d, J0
11.0 Hz, 1 H, PhCH2), 4.78 (d, J08.0 Hz, 1 H, H-1E), 4.76 (d,
J07.5 Hz, 1 H, H-1C), 4.70–4.60 (2 d, J011.5 Hz each,
PhCH2), 4.49–4.41 (2 d, J011.0 Hz each, PhCH2), 4.20 (d,
J03.0 Hz, 1 H, H-4D), 4.17 (d, J03.5 Hz, 1 H, H-4C), 4.12–
4.10 (m, 1 H, H-6aE), 4.07–4.04 (m, 1 H, H-6bE), 4.03 (br s, 1
H, H-5C), 3.99 (dd, J010.5, 3.0 Hz, 1 H, H-3D), 3.95–3.88 (m,
2 H, H-5E, H-6aC), 3.79–3.76 (m, 2 H, H-2C, H-2D), 3.69 (s, 3
H, OCH3), 3.60–3.55 (m, 3 H, H-5D, H-6aD, H-6bC), 3.44 (dd,
J010.0, 3.0 Hz, 1 H, H-3C), 3.37–3.35 (m, 1 H, H-6bD), 2.06,
2.00, 1.93, 1.90 (4 s, 12 H, 4 COCH3); 13C NMR (125 MHz,
CDCl3): δ 170.4, 170.3, 170.2, 169.5 (4 COCH3), 155.4–
114.5 (Ar-C), 103.4 (C-1E), 103.4 (C-1C), 100.5 (PhCH),
p-Methoxyphenyl (4,6-O-benzylidene-2-azido-2-deoxy-α-D-
galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-galacto-
pyranoside (9) A solution of compound 8 (1.2 g,
1.38 mmol) in 0.1 M CH3ONa in CH3OH (20 mL) was
allowed to stir at room temperature for 2 h. The reaction
mixture was neutralized with Dowex 50 W X8 (H+) resin,
filtered and concentrated under reduced pressure. To a so-
lution of the deacetylated product in anhydrous CH3CN-
DMF (10 mL; 1:1 v/v) was added benzaldehyde dimethyl
acetal (0.4 mL, 2.66 mmol) followed by HClO4-SiO2
(50 mg) and the reaction mixture was allowed to stir at
room temperature for 5 h. The reaction mixture was filtered
and the solvents were removed under reduced pressure to
give the crude product, which was purified over SiO2 using
hexane-EtOAc (5:1) as eluant to give pure compound 9
25
(880.0 mg, 77 %). Colourless oil; [α]D +86.8 (c 1.2,
CHCl3); IR (neat): 3336, 2926, 2121, 1737, 1716, 1514,
1467, 1226, 1170, 1067, 756, 699 cm−1 1H NMR
;
(500 MHz, CDCl3): δ 7.38–7.17 (m, 20 H, Ar-H), 6.97 (d,
J09.0 Hz, 2 H, Ar-H), 6.72 (d, J09.0 Hz, 2 H, Ar-H), 5.34
(s, 1 H, PhCH), 4.97 (d, J03.5 Hz, 1 H, H-1D), 4.95 (d, J0

186
Glycoconj J (2012) 29:181–188
99.6 (C-1D), 80.6 (C-3C), 78.5 (C-2C), 76.6 (C-4D), 75.6 (C-
3D), 75.0 (PhCH2), 73.7 (PhCH2), 73.2 (C-4C), 73.1 (C-5D),
72.7 (PhCH2), 71.2 (C-3E), 70.8 (C-5E), 69.0 (C-6C), 68.8 (C-
2E), 67.0 (C-4E), 66.9 (C-6D), 62.9 (C-5C), 61.5 (C-6E), 59.4
(C-2D), 55.6 (OCH3), 20.9, 20.8, 20.7, 20.6 (4 COCH3); ESI-
MS: 1184.4 [M+Na]+; Anal. Calcd. for C61H67N3O20
(1161.43): C, 63.04; H, 5.81; found: C, 62.82; H, 6.04.
CAN (1.0 g, 1.82 mmol) and the reaction mixture was
allowed to stir at room temperature for 2 h. The reaction
mixture was extracted with EtOAc (50 mL). The organic
layer was successively washed with satd. NaHCO3 and
water, dried (Na2SO4) and concentrated. The crude product
was passed through a short pad of SiO2 using hexane-
EtOAc (3:1) to give trisaccharide hemiacetal derivative. To
a solution of the hemiacetal derivative in anhydrous CH2Cl2
(5 mL) was added CCl3CN (0.4 mL, 3.98 mmol) and cooled
to −20 °C. To the cooled reaction mixture was added DBU
(10 μL) and the reaction mixture was stirred at same tem-
perature for 1 h. The solvents were removed under reduced
pressure and the crude product was purified over SiO2 using
hexane-EtOAc (6:1) as eluant to give pure compound 12
(500.0 mg, 74 %), which was used immediately for the next
step without further characterization.
p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyra-
nosyl)-(1→3)-(4,6-di-O-acetyl-2-azido-2-deoxy-α-D-galac-
topyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-galactopyrano-
side (11) To a solution of compound 10 (850.0 mg,
0.73 mmol) in acetic anhydride (2 mL) was added HClO4-
SiO2 (50.0 mg) and the reaction mixture was allowed to stir
at room temperature for 30 min. The reaction mixture was
filtered and washed with EtOAc (25 mL). The organic layer
was washed with satd. NaHCO3 and water, dried (Na2SO4)
and concentrated under reduced pressure. The crude product
was passed through a short pad of SiO2 using hexane-
EtOAc (3:1) to give pure compound 11 (675.0 mg, 80 %).
p-Methoxyphenyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyra-
nosyl)-(1→3)-(4,6-di-O-acetyl-2-azido-2-deoxy-α-D-galac-
topyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-α-D-galactopyra-
nosyl-(1→3)-(2-O-benzyl-4,6-O-benzylidene-α-D-galacto-
pyranosyl)-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthali-
mido-β-D-glucopyranoside (13) A solution of compound 7
(320.0 mg, 0.38 mmol) and compound 12 (500.0 mg,
0.42 mmol) in anhydrous CH2Cl2-Et2O (5 mL, 3:1 v/v)
was cooled to −10 °C. 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 and washed with CH2Cl2 (50 mL). The
combined organic layer was washed with satd. NaHCO3 and
water, dried (Na2SO4) and concentrated. The crude product
was purified over SiO2 using hexane-EtOAc (4:1) as eluant
to give pure compound 13 (485.0 mg, 68 %). Colourless oil;
25
Colourless oil; [α]D +58 (c 1.2, CHCl3); IR (neat): 3484,
3036, 2957, 2931, 1757, 1508, 1378, 1244, 1179, 1093,
1
977, 698 cm−1; H NMR (500 MHz, CDCl3): δ 7.29–7.17
(m, 15 H, Ar-H), 6.90 (d, J09.0 Hz, 2 H, Ar-H), 6.71 (d, J0
9.0 Hz, 2 H, Ar-H), 5.41 (d, J02.0 Hz, 1 H, H-4E), 5.30 (d,
J03.0 Hz, 1 H, H-4D), 5.12 (dd, J08.0 Hz each, 1 H, H-2E),
4.96 (dd, J09.5, 3.0 Hz, 1 H, H-3E), 4.94 (d, J011.0 Hz, 1
H, PhCH2), 4.91 (d, J03.5 Hz, 1 H, H-1D), 4.80 (d, J0
11.0 Hz, 1 H, PhCH2), 4.72 (d, J07.5 Hz, 1 H, H-1C), 4.70
(d, J011.0 Hz, 1 H, PhCH2), 4.78 (d, J08.0 Hz, 1 H, H-1E),
4.63 (d, J011.0 Hz, 1 H, PhCH2), 4.48–4.45 (m, 1 H, H-5D),
4.43 (br s, 2 H, PhCH2), 4.10–4.00 (m, 4 H, H-3D, H-4C, H-
6abE), 3.90–3.85 (m, 3 H, H-6aC, H-6abD), 3.75 (dd, J0
7.5 Hz each, 1 H, H-2C), 3.68 (s, 3 H, OCH3), 3.59 (dd,
J09.5, 3.0 Hz, 1 H, H-2D), 3.57–3.50 (m, 3 H, H-5C, H-5E,
H-6bC), 3.39 (dd, J010.0, 3.0 Hz, 1 H, H-3C), 2.09, 2.00,
1.95, 1.90, 1.87 (6 s, 18 H, 6 COCH3); 13C NMR (125 MHz,
CDCl3): δ 170.4, 170.3, 170.2, 170.1, 169.7, 169.5 (6
COCH3), 155.3–114.5 (Ar-C), 103.2 (C-1C), 100.6 (C-1E),
98.8 (C-1D), 79.8 (C-3C), 78.6 (C-2C), 75.1 (C-5E), 75.0
(PhCH2), 73.9 (C-3D), 73.6 (PhCH2), 73.1 (PhCH2), 73.0
(C-5C), 71.0 (C-3E), 70.9 (C-4C), 69.3 (C-4E), 68.3 (C-2E),
67.0 (C-6C), 66.8 (C-5D), 66.7 (C-4D), 61.5 (C-6D), 61.0 (C-
6E), 60.2 (C-2D), 55.6 (OCH3), 21.0, 20.7 (2 C), 20.6 (2 C),
20.5 (6 COCH3); ESI-MS: 1180.4 [M+Na]+; Anal. Calcd.
for C58H67N3O22 (1157.42): C, 60.15; H, 5.83; found: C,
60.00; H, 6.00.
25
[α]D +92 (c 1.2, CHCl3); IR (neat): 3022, 2365, 1746,
1653, 1218, 769, 677 cm−1; 1H NMR (500 MHz, CDCl3): δ
7.91–6.92 (m, 34 H, Ar-H), 6.87 (d, J09.0 Hz, 2 H, Ar-H),
6.76 (d, J09.0 Hz, 2 H, Ar-H), 6.76 (d, J08.5 Hz, 1 H, H-
1A), 5.48 (d, J03.5 Hz, 1 H, H-1B), 5.44 (d, J03.0 Hz, 1 H,
H-4D), 5.39 (br s, 1 H, H-4E), 5.38 (s, 1 H, PhCH), 5.26 (d,
J03.0 Hz, 1 H, H-1C), 5.22 (dd, J08.0 Hz each, 1 H, H-2E),
5.18 (s, 1 H, PhCH), 5.08 (d, J03.5 Hz, 1 H, H-1D), 5.02
(dd, J010.0, 3.0 Hz, 1 H, H-3E), 4.92 (t, J09.0 Hz, 1 H
each, H-3A), 4.60–4.43 (m, 8 H, H-2A, H-5D, PhCH2), 4.52
(d, J08.0 Hz, 1 H, H-1E), 4.33–4.22 (m, 3 H, H-5A,
PhCH2), 4.08–3.96 (m, 4 H, H-4C, H-6abE, H-6aD), 3.95–
3.88 (m, 4 H, H-3B, H-3D, H-5C, H-6bD), 3.86–3.67 (m, 8 H,
H-2B, H-3C, H-4A, H-4B, H-5E, H-6aA, H-6abC), 3.64 (s, 3 H,
OCH3), 3.61–3.58 (m, 1 H, H-2C), 3.52–3.45 (m, 2 H, H-2D,
H-6bA), 3.38–3.33 (m, 1 H, H-6aB), 2.95–2.88 (m, 2 H, H-
5B, H-6bB), 2.06, 2.00, 1.98, 1.90, 1.89, 1.82 (6 s, 18 H, 6
COCH3); 13C NMR (125 MHz, CDCl3): δ 170.3 (2 C),
170.2 (2 C), 169.6, 169.5 (6 COCH3), 155.6–114.5 (Ar-
C), 101.8 (PhCH), 101.2 (PhCH), 100.7 (JC-1/H-1 0
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→3)-(4,6-
di-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-α/β-D-galactopyranosyl trichlor-
oacetimidate (12) To a solution of compound 11 (650.0 mg,
0.56 mmol) in CH3CN-H2O (15 mL; 1:1 v/v) was added

Glycoconj J (2012) 29:181–188
187
158.0 Hz) (C-1E), 98.3 (JC-1/H-10170.0 Hz) (C-1B), 98.1
(JC-1/H-10160.0 Hz) (C-1A), 98.0 (JC-1/H-10169.0 Hz) (C-
1D), 91.7 (JC-1/H-10172.0 Hz) (C-1C), 82.2 (C-4A), 76.0 (C-
4B), 75.5 (C-3C), 75.0 (C-2B), 73.8 (C-3A), 73.1 (PhCH2),
72.0 (C-5C), 71.8 (PhCH2), 71.7 (C-3D), 70.9 (C-5E), 70.6
(2 C, C-3E, C-5A), 70.5 (PhCH2), 70.4 (C-3B), 69.1 (C-4C),
68.9 (C-2E), 68.8 (C-4D), 68.7 (2 C, C-6B, PhCH2), 66.7 (C-
2C), 66.6 (C-6C), 66.4 (C-5D), 66.1 (C-4E), 62.6 (C-5B), 61.5
(C-6A), 60.9 (2 C, C-6D, C-6E), 60.1 (C-2D), 55.6 (OCH3),
55.2 (C-2A), 20.8, 20.7, 20.6 (2 C), 20.5 (2 C) (6 COCH3);
MALDI-TOF-MS: 1899.6 [M+Na]+; Anal. Calcd. for
C99H104N4O33 (1876.66): C, 63.32; H, 5.58; found: C,
63.10; H, 5.84.
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 concentrat-
ed. A solution of the crude product in 0.1 M CH3ONa in
CH3OH (5 mL) was allowed to stir at room temperature for
3 h, neutralized with Dowex 50 W X8 (H+) resin and then
treated with Dowex 50 W X8 (Na+) resin, filtered and
evaporated to dryness. The sodium salt of the pentasacchar-
ide was purified through a Sephadex® LH-20 column using
CH3OH-H2O (3:1) as eluant to give pure compound 1
25
(130.0 mg, 51 %) as white powder. [α]D +67 (c 1.2,
CH3OH); IR (KBr): 2364, 2343, 1727, 1646, 1567, 1382,
1074, 838, 770, 676 cm−1; 1H NMR (500 MHz, CD3OD): δ
6.79 (d, J09.0 Hz, 2 H, Ar-H), 6.73 (d, J09.0 Hz, 2 H, Ar-
H), 5.15 (br s, 1 H, H-1C), 4.90 (br s, 2 H, H-1B, H-1D),
4.80–4.78 (m, 1 H, H-1A), 4.32 (d, J08.0 Hz, 2 H, H-1E),
4.27–3.89 (m, 7 H, H-2A, H-2D, H-4A, H-4C, H-4D, H-4E,
H-5C), 3.90–3.72 (m, 8 H, H-2C, H-3A, H-3B, H-3C, H-3D,
p-Methoxyphenyl (β-D-galactopyranosyl)-(1→3)-(2-acet-
amido-2-deoxy-α-D-galactopyranosyl)-(1→4)-(sodium α-
D-galactopyranosyl uronate)-(1→3)-(α-D-galactopyrano-
syl)-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside (1)
To a solution of compound 13 (450.0 mg, 0.24 mmol) in
CH3OH (8 mL) was added 20 % Pd(OH)2-C (100 mg) and
the reaction mixture was allowed to stir under a positive
pressure of hydrogen at room temperature for 4 h. The
reaction mixture was filtered through a Celite® bed and
concentrated to the one third of the reaction volume. To
the methanolic solution of the selectively hydrogenated
product was added acetic anhydride (1 mL) and the reaction
mixture was kept at room temperature for 1 h. The solvents
were removed under reduced pressure to give N-acetylated
and debenzylated product. To a solution of the pentasac-
charide tetraol derivative in CH2Cl2 (20 mL) and H2O
(5 mL) were successively added aq. NaBr (3 mL; 1 M),
aq. TBAB (5 mL; 1 M), TEMPO (150.0 mg, 0.96 mmol),
satd. NaHCO3 (15 mL) and 4 % aq. NaOCl (15 mL) and the
reaction mixture was allowed to stir at 5 °C for 3 h and
neutralized with 1 N HCl. To the reaction mixture were
added tert-butanol (20 mL), 2-methyl-but-2-ene (20 mL; 2
M solution in THF), aq. NaClO2 (2.0 g/10 mL) and aq.
NaH2PO4 (2.0 g/10 mL) and the reaction mixture was
allowed to stir at room temperature for 3 h. The reaction
mixture was diluted with satd. aq. NaH2PO4 and extracted
with CH2Cl2 (100 mL). The organic layer was washed with
water, dried (Na2SO4) and concentrated to dryness to give
the crude product, which was passed through a short pad of
SiO2. To a solution of the sodium salt of the oxidized
product in C2H5OH (10 mL) was added hydrazine mono-
hydrate (0.2 mL) and the reaction was allowed to stir at 80 °
C for 7 h. The solvents were removed under reduced pres-
sure and the crude product was dissolved in acetic anhydride
and pyridine (2 mL, 1:1 v/v) and kept at room temperature
for 2 h. The solvents were removed under reduced pressure
and the crude product was passed through a short pad of
SiO2. To a solution of the acetylated product in CH3OH
(5 mL) was added 20 % Pd(OH)2-C (100 mg) and the
H-5B, H-6abE), 3.70–3.54 (m, 8 H, H-2E, H-6abA, H-6abD
,
OCH3), 3.52–3.30 (m, 7 H, H-2B, H-3E, H-5A, H-4D, H-5E,
H-6abB), 1.92 (br s, 6 H, 2 COCH3); 13C NMR (125 MHz,
CD3OD): δ 174.6 (COONa), 172.7, 172.6 (2 COCH3),
155.2–114.1 (Ar-C), 104.9 (C-1E), 102.6 (C-1A), 99.1 (2
C, C-1B, C-1D), 96.1 (C-1C), 78.8 (C-4C), 77.5 (C-3A), 76.5
(C-3D), 76.3 (C-3B), 75.3 (2 C, C-5A, C-5E), 73.3 (2 C, C-
3E, C-4D), 71.3 (2 C, C-2E, C-4A), 71.1 (C-5D), 69.7 (C-4E),
68.9 (2 C, C-2C, C-3C), 68.6 (C-2B), 67.7 (C-5B), 65.2 (C-
5C), 63.0 (C-4B), 61.2 (3 C, C-6A, C-6B, C-6E), 60.2 (C-6D),
55.8 (C-2D), 55.4 (C-2A), 54.7 (OCH3), 21.5 (2 C, 2
COCH3); ESI-MS: 1053.3 [M+1]+; Anal. Calcd. for
C41H61N2NaO28 (1052.33): C, 46.77; H, 5.84; found: C,
47.0; H, 6.12.
Acknowledgments A. S. thanks CSIR, New Delhi for providing a
Senior Research Fellowship. This work was supported by the Depart-
ment of Science and Technology (DST), New Delhi (Project no. SR/
S1/OC-83/2010).
References
1. Karch, H., Tarr, P., Bielaszewska, M.: Enterohaemorrhagic Escher-
ichia coli in human medicine. Int. J. Med. Microbiol. 295, 405–418
(2005)
2. Russmann, H., Kothe, E., Schmidth, H., Franke, S., Harmsen, D.,
Caprioli, A., Karch, H.: Genotyping of Shiga-like toxin genes in
non-O157 Escherichia coli strains associated with haemolytic
uraemic syndrome. J. Med. Microbiol. 42, 404–410 (1995)
3. Bower, J.R.: Food-borne diseases: Shiga toxin producing E. coli
(STEC). Pediatr. J. Infect. Dis. 18, 909–910 (1999)
4. Ludwig, K., Sarkim, V., Bitzan, M., Karmali, M.A., Bobrowski,
C., Ruder, H., Laufs, R., Sobottka, I., Petric, M., Karch, H.,
Muller-Wiefel, D.E.: Shiga toxin-producing Escherichia coli infec-
tion and antibodies against Stx2 and Stx1 in household contacts of
children with enteropathic hemolytic-uremic syndrome. J. Clin.
Microbiol. 40, 1773–1782 (2002)

188
5. Kaper, J.B.: Enterohemorrhagic Escherichia coli. Curr. Opin.
Glycoconj J (2012) 29:181–188
20. Konradsson, P., Udodong, U.E., Fraser-Reid, B.: Iodonium pro-
moted reactions of disarmed thioglycosides. Tetrahedron Lett. 31,
4313–4316 (1990)
21. Du, Y., Wei, G., Cheng, S., Hua, Y., Linhardt, R.J.: HClO4-SiO2
catalyzed glycosylation using trichloroacetimidates as glycosyl
donors. Tetrahedron Lett. 47, 307–310 (2006)
22. Sau, A., Misra, A.K.: Environmentally benign preparation of ben-
zylidene acetal of carbohydrate derivatives in PEG 600. J. Carbo-
hydr. Chem. 30, 41–46 (2011)
23. Mukherjee, C., Misra, A.K.: Glycosylation and pyranose-furanose
isomerization of carbohydrates using HClO4-SiO2: synthesis of
oligosaccharides containing galactofuranose. Synthesis 683–692
(2007)
Microbiol. 1, 103–108 (1998)
6. Boyce, T.G., Swerdlow, D.L., Griffin, P.M.: Escherichia coli
O157:H7 and the hemolytic uremic syndrome. N. Engl. J. Med.
333, 364–368 (1995)
7. Stenutz, R., Weintraub, A., Widmalm, G.: The structures of
Escherichia coli O-polysaccharide antigens. FEMS Microbiol.
Rev. 30, 382–403 (2006)
8. Roy, R.: New trends in carbohydrate based vaccines. Drug Discov.
Today Technol. 1, 327–336 (2004). and references cited therein
9. Borman, S.A.: Carbohydrate vaccines: novel chemical and enzy-
matic oligosaccharide synthesis techniques could lead to a new
generation of carbohydrate-based vaccine agents. Chem. Eng.
News. 82, 31–35 (2004)
10. Doshi, G.M., Shanbhag, P.P., Aggarwal, G.V., Shahare, M.D.,
Martis, E.A.: Carbohydrate vaccines- A burgeoning field of Gly-
comics. J. Appl. Pharm. Sci. 1, 17–22 (2011)
24. Agnihotri, G., Misra, A.K.: Mild and efficient method for the
cleavage of benzylidene acetals using HClO4 SiO2 and direct
conversion of acetals to acetates. Tetrahedron Lett. 47, 3653–
3658 (2006)
11. Parolis, H., Parolis, L.A.S.: The structure of the O-specific poly-
saccharide from Escherichia coli O113 lipopolysaccharide. Carbo-
hydr. Res. 267, 263–269 (1995)
12. Guchhait, G., Misra, A.K.: Convergent synthesis of a common
pentasaccharide corresponding to the O-antigen of Escherichia
coli O168 and Shigella dysenteriae type 4. Glyconjugate J. 28,
11–19 (2011)
13. Panchadhayee, R., Misra, A.K.: First synthesis of pentasaccharide
repeating unit of the O-antigenic polysaccharide from enterohae-
morrhagic Escherichia coli O48:H21. Tetrahedron: Asymm. 20,
1550–1555 (2009)
14. Kawai, Y., Ando, H., Ozeki, H., Koketsu, M., Ishihara, H.: A facile
method for β-selenoglycoside synthesis using β-p-methylbenzoyl
selenoglycoside as the selenating unit. Org. Lett. 7, 4653–4656
(2005)
15. Grundler, G., Schmidt, R.R.: Glycosyl imidates. 13. Application of
the trichloroacetimidate procedure to 2-azidoglucose and 2-
azidogalactose derivatives. Liebigs Annal. Chem. 11, 1826–1847
(1984)
16. Agnihotri, G., Tiwari, P., Misra, A.K.: Onepot synthesis of per-
O acetylated thioglycosides from unprotected reducing sugars.
Carbohydr. Res. 340, 1393–1396 (2005)
17. Bhattacharyya, S., Magnusson, B.G., Wellmer, U., Nilsson, U.J.:
The p-methoxybenzyl ether as an in situ-removable carbohydrate
protecting group: a simple one-pot synthesis of the globotetraose
tetrasaccharide. J. Chem. Soc. Perkin Trans. 1, 886–890 (2001)
18. Lemieux, R.U., Ratcliffe, R.M.: The azidonitration of tri-O-acetyl-
D-galactal. Can. J. Chem. 57, 1244–1251 (1979)
19. Veeneman, G.H., van Leeuwen, S.H., van Boom, J.H.: Iodonium
ion promoted reactions at the anomeric centre. II An efficient
thioglycoside mediated approach toward the formation of 1,2-trans
linked glycosides and glycosidic esters. Tetrahedron Lett. 31,
1331–1334 (1990)
25. Werz, D.B., Seeberger, P.H.: Total Synthesis of Antigen Bacillus
Anthracis Tetrasaccharide-Creation of an Anthrax Vaccine Candi-
date. Angew. Chem. Int. Ed. 44, 6315–6318 (2005)
26. Han, X.-B., Jiang, Z.-H., Schmidt, R.R.: Glycosyl Imidates, 61.–
Synthesis of the Hexasaccharide Moiety of the Saponin Holotoxin
A. Liebigs Annal. Chem. 853–858 (1993)
27. Bock, K., Lundt, I., Pedersen, C.: Assignment of anomeric struc-
ture to carbohydrates through geminal carbon-13-proton coupling
constants. Tetrahedron Lett. 13, 1037–1040 (1973)
28. Crich, D., Li, H.: Synthesis of the Salmonella type E1 core trisac-
charide as a probe for the generality of 1-(benzenesulfinyl)piperi-
dine/triflic anhydride combination for glycosidic bond formation
from thioglycoside. J. Org. Chem. 67, 4640–4646 (2002)
29. Misra, A.K., Ding, Y., Lowe, J.B., Hindsgaul, O.: A concise
synthesis of the 6-O and 6′-O-sulfated analogues of the sialyl
Lewis X tetrasaccharide. Bioorg. Med. Chem. Lett. 10, 1505–
1509 (2000)
30. Huang, L., Teumelsan, N., Huang, X.: A facile method for oxida-
tion of primary alcohols to carboxylic acids and its application in
glycosaminoglycan syntheses. Chem. Eur. J. 12, 5246–5252
(2006)
31. Anelli, P.L., Biffi, C., Montanari, F., Quici, S.: Fast and selective
oxidation of primary alcohols to aldehydes or to carboxylic acids
and of secondary alcohols to ketones mediated by oxoammonium
salts under two-phase conditions. J. Org. Chem. 52, 2559–2562
(1987)
32. Panchadhayee, R., Misra, A.K.: Convergent synthesis of the pen-
tasaccharide repeating unit of the O-antigen of Shigella boydii type
14. Tetrahedron: Asymm. 22, 1390–1394 (2011)
33. Chakraborti, A.K., Gulhane, R.: Perchloric acid adsorbed on silica
gel as a new, highly efficient and versatile catalyst for acetylation
of phenols, thiols, alcohols and amines. Chem. Commun. 1896–
1897 (2003)