Synthesis of Tri- and disaccharide fragments related to the O-Antigen of enteropathogenic Escherichia Coli O158

Article abstract of DOI:10.1080/07328300903477804

Chemical synthesis of a tri- and a disaccharide related to the O-antigen of enteropathogenic E. coli O158 was achieved in high yield. In the disaccharide (2), one D-galactosamine unit is present in 1,2-cis-linked and the other is in the trans-orientation,

Full text of DOI:10.1080/07328300903477804

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Synthesis of Tri- and Disaccharide
Fragments Related to the O-Antigen of
Enteropathogenic Escherichia Coli O158
Rajib Panchadhayee a & Anup Kumar Misra a
a Division of Molecular Medicine , Bose Institute , A.J.C. Bose
Centenary Campus, P-1/12, C.I.T. Scheme VII-M, Kolkata, 700054,
India
Published online: 04 Jan 2010.
To cite this article: Rajib Panchadhayee & Anup Kumar Misra (2010) Synthesis of Tri- and Disaccharide
Fragments Related to the O-Antigen of Enteropathogenic Escherichia Coli O158, Journal of
Carbohydrate Chemistry, 29:1, 39-50, DOI: 10.1080/07328300903477804
To link to this article: http://dx.doi.org/10.1080/07328300903477804
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Journal of Carbohydrate Chemistry, 29:39–50, 2010
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ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300903477804
Synthesis of Tri- and
Disaccharide Fragments
Related to the O-Antigen of
Enteropathogenic Escherichia
Coli O158
Rajib Panchadhayee and Anup Kumar Misra
Division of Molecular Medicine, Bose Institute, A.J.C. Bose Centenary Campus,
P-1/12, C.I.T. Scheme VII-M, Kolkata-700054, India
Chemical synthesis of a tri- and a disaccharide related to the O-antigen of en-
teropathogenic E. coli O158 was achieved in high yield. In the disaccharide (2), one D-
galactosamine unit is present in 1,2-cis-linked and the other is in the trans-orientation,
and both of them were prepared from the same intermediate by tuning the reaction
solvent. Yields were considerably high in all steps.
Keywords Trisaccharide; Disaccharide, E. coli; Glycosylation; Enteropathogenic
INTRODUCTION
Escherichia coli (E. coli) bacteria exist as harmless species to life-threatening
microorganisms. In general, it is a nonpathogenic member of the human
colonic flora. However, certain species of E. coli have acquired virulence fac-
tors and behave as an opportunistic pathogen responsible for several intesti-
nal and urinary diseases in humans and animals. Three most frequent clinical
syndromes caused by E. coli are (a) diarrhea, (b) urinary tract infections, and
(c) sepsis and meningitis.[1] E. coli causing diarrhea are divided in six cate-
gories depending on the type of disease[2]: (1) enteropathogenic E. coli (EPEC),
(2) enterotoxigenic E. coli (ETEC), (3) enteroinvasive E. coli (EIEC), (4)
Received September 2, 2009; accepted November 10, 2009.
Address correspondence to Anup Kumar Misra, Division of Molecular Medicine, Bose
Institute, A.J.C. Bose Centenary Campus, P-1/12, C.I.T. Scheme VII-M, Kolkata 700054,
India. E-mail: akmisra69@rediffmail.com
39

R. Panchadhayee & A.K. Misra
40
α-D-Glcp
1
↓
6
→3)-α-D-GalpNAc-(1→3)-β-D-GalpNAc-(1→4)-α-D-Glcp-(1→
3
↑
1
α-L-Rhap
Figure 1: Structure of the pentasaccharide repeating unit of the O-antigen from the
enteropathogenic Escherichia coli O158.
enterohaemorrhagic E. coli (EHEC), (5) enteroaggregative E. coli (EAEC), and
(6) diffusely adherent E. coli (DAEC).
The enteropathogenic E. coli (EPEC) strains are known to be associated
with various kinds of diarrhea in infants and are a cause of illness and death
among children in the developing countries.[3] The O-antigen or endotoxins are
the responsible virulence factor of the enteropathogenic E. coli strains, which
mediate their interactions with the host at the initial stage of bacterial infec-
tion.[4] The structure of the pentasaccharide O-antigen of the enteropathogenic
E. coli strain O158 has been reported by Datta et al. (Fig. 1).[5] The structure
of the pentasaccharide repeating unit is unique in nature as it contains two
D-glucopyranosyl and one L-rhamnopyranosyl moieties α-glycosidically linked
and a disaccharide branch in which one D-galactosamine unit exists as α-
glycosidically linked and the other one is β-glycosidically linked. In the recent
past, chemical synthesis of immunodominant oligosaccharides has gained con-
siderable interest.[6] In order to understand the relationship between structure
and immunochemical specificity of the O-antigen, we decided to prepare a di-
and a trisaccharide fragment related to the pentasaccharide repeating unit
of the O-antigen of E. coli O158. We herein describe concise chemical synthe-
sis of a tri- and a disaccharide (1 and 2) as their methyl glycoside and 2-(4-
methoxyphenoxy) ethyl glycoside, respectively (Fig. 2).
RESULTS AND DISCUSSION
Trisaccharide 1 and disaccharide 2 were synthesized from a series of suit-
ably protected monosaccharide intermediates by regio- and stereoselective
glycosylations. The functionalized monosaccharide intermediates 3,[7] 4,[8] and
5[9] were prepared from the commercially available monosaccharides using
literature-reported reaction conditions. Iodonium ion promoted stereoselective
glycosylation of compound 3 with thioglycoside derivative 4 in the presence of a
N-iodosuccinimide (NIS)-trimethylsilyl trifluoromethanesulfonate (TMSOTf)
combination,[10] which furnished disaccharide derivative 6 in 88% yield, which

Synthesis of Escherichia coli O158
41
OH
O
SEt
Ph
O
O
HO
HO
O
C
O
AcO
HO
HO
BnO
OCH3
AcO
OAc
O
3
4
O
HO
A
O
OBn
O
HO
BnO
BnO
OCH3
B
O
SEt
HO
BnO
5
HO
OH
1
AcO OAc
O
HO OH
O
B
HO OH
AcO
HO
N3
AcHN
O
A
OC(NH)CCl3
O
OMPE
11
2
NHAc
OCH3
O
OCH3
O
HO
MPE:
12
Figure 2: Structures of the synthesized tri- and disaccharide fragments corresponding to the
O-antigen of enteropathogenic Escherichia coli O158.
was transformed to disaccharide derivative 7 under a one-pot deacetylation-
benzylation[11] reaction condition in 90% yield. Removal of the benzylidene ac-
etal[12] of compound 7 in the presence of HClO4-SiO2
resulted in the disac-
[13]
charide diol derivative 8 in 79% yield. Regio- and stereoselective glycosylation
of compound 8 with thioglycoside donor 5 in the presence of NIS-TMSOTf fur-
nished trisaccharide derivative 9 in 80% yield. Appearance of a signal at δ
1
4.83 (d, J = 3.8 Hz) and 98.4 in the H and 13C NMR spectrum, respectively,
confirmed the formation of compound 9 with the desired stereo outcome. Hy-
drogenation over Pearlman’s catalyst furnished target trisaccharide 1 in 68%.
It is worth mentioning that in compound 1, all monosaccharide units are α-
glycosidically linked (Scheme 1).
In a separate experiment, disaccharide 2 was synthesized as a 2-(4-
methoxyphenoxy) ethyl glycoside (2). Following literature-reported proto-
col, 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl trichloroacetimi-
date (11)[14] was prepared from tri-O-acetyl-D-galactal (10) in three steps.
Compound 11 was stereoselectively glycosylated with 2-(4-methoxyphenoxy)
ethanol (12)[15] in the presence of TMSOTf in CH3CN[16] to give compound 13
in 78% yield. Exclusive formation of 1,2-trans glycoside was achieved by ex-
ploiting the nitrile effect of the solvent. Sequential deacetylation and benzyli-
dene acetal formation of compound 13 furnished compound 14 in 87% yield.
Another α-selective glycosylation of compound 13 with glycosyl donor 11 in the
presence of TMSOTf in methylene chloride[17] furnished disaccharide deriva-
tive 15 in 75% yield. Finally, hydrogenolysis[18] of disaccharide derivative 15

R. Panchadhayee & A.K. Misra
42
SEt
Ph
O
O
O
Ph
O
A
O
a
O
O
O
c
AcO
+
BnO
OCH3
HO
AcO
BnO
B O
OAc
RO
OCH3
4
3
RO
OR
6: R = Ac
7: R = Bn
b
OBn
O
BnO
C
OH
O
BnO
BnO
HO
A
d
O
5
a
O
1
BnO
OCH3
O
HO
A
B O
O
BnO
BnO
BnO
OCH3
OBn
B O
BnO
BnO
8
OBn
9
Scheme 1: Reagents: (a) N-iodosuccinimide, TMSOTf, CH2Cl2, MS 4Å, −40◦C, 1 h, 88% for 6
and 80% for 9; (b) benzyl bromide, NaOH, TBAB, THF, rt, 6 h, 90%; (c) HClO4-SiO2, CH3CN, H2O,
rt, 20 min, 79%; (d) H2, 20% Pd(OH)2-C, CH3OH, rt, 24 h, 68%.
followed by N-acetylation and O-deacetylation afforded target disaccharide 2
as its 2-(4-methoxyphenoxy) ethyl glycoside in 72% yield (Scheme 2).
In summary, a tri- and a disaccharide related to the O-antigen of E. coli
O158 were synthesized in excellent yield as methyl and 2-(4-methoxyphenoxy)
ethyl glycoside, respectively. All glycosylation and protecting group manipu-
lation steps were high yielding and reproducible for scale-up preparation. All
AcO OAc
OAc
O
AcO
AcO
OMp
HO
O
12
a
AcO
b
N3
10
OAc
OC(NH)CCl3
11
Ph
AcO
AcO
O
Mp: 4-Methoxyphenyl
OMp
O
c
O
OMp
O
O
HO
O
N3
N3
13
14
OAc
O
AcO
Ph
B
O
AcO
e
d
O
2
11
14
+
N3
O
O
A
OMp
O
N3
15
Scheme 2: Reagents: (a) Ref. [14]; (b) TMSOTf, CH3CN, −20◦C, 1 h, 78%; (c) (i) 0.1 N CH3ONa,
CH3OH, rt, 3 h; (ii) PhCH(OCH3)2, p-TsOH, CH3CN, rt, 12 h, 87%; (d) TMSOTf, CH2Cl2, −20◦C, 1
h, 75%; (e) (i) H2, 20% Pd(OH)2-C, CH3OH, rt, 12 h; (ii) acetic anhydride, pyridine, rt, 2 h; (iii)
0.1 N CH3ONa, CH3OH, rt, 6 h, 72%.

Synthesis of Escherichia coli O158
43
monosaccharide moieties are α-linked in trisaccharide 1. The disaccharide 2
was prepared from a single intermediate having a nonparticipating group at
the C-2 position employing the solvent effect on the stereo outcome of the gly-
cosylation.
EXPERIMENTAL
General Procedure
All reactions were monitored by thin layer chromatography using silica
gel-coated TLC plates. The spots on TLC were visualized by warming ceric
sulphate (2% Ce(SO4)2 in 2N H2SO4)-sprayed plates on a hot plate. Silica gel
1
230–400 mesh was used for column chromatography. H and 13C NMR, 2D
COSY, and HMQC spectra were recorded on a Bruker Avance DRX 500 MHz
using CDCl3 and CD3OD as solvents and TMS as internal reference unless
stated otherwise. Chemical shift values are expressed in δ ppm. ESI-MS was
recorded on a Micromass Quattro II triple quadrupole mass spectrometer. El-
ementary analysis was carried out on a Carlo ERBA-1108 analyzer. Optical
rotations were measured at 25◦C on a Perkin Elmer 341 polarimeter. Com-
mercially available grades of organic solvents of adequate purity were used in
many reactions.
Methyl (2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-(1→3)-2-O-
benzyl-4,6-O-benzylidene-α-D-glucopyranoside (6)
To a solution of compound 3 (1.5 g, 4.03 mmol) and compound 4 (1.6 g, 4.78
˚
mmol) in anhydrous CH2Cl2 (20 mL) was added MS 4A (2 g) and the reaction
mixture was allowed to stir at rt under argon for 1 h. The reaction mixture was
cooled to −40◦C; to the cold reaction mixture were added N-iodosuccinimide
(NIS; 1.3 g, 5.77 mmol) and TMSOTf (25 µL), and the reaction mixture was
allowed to stir at the same temperature for 1 h. The reaction mixture was fil-
tered through a Celite bed and washed with CH2Cl2 (100 mL). The organic
layer was successively washed with 5% Na2S2O3, satd. NaHCO3, and H2O;
dried (Na2SO4); and concentrated. The crude product was purified over SiO2
using hexane-EtOAc (6:1) as eluant to give pure 6 (2.3 g, 88%). Yellow oil; IR
(neat): 2937, 1748, 1373, 1248, 1224, 1137, 1088, 1048, 986, 750, 699 cm−1
;
1H NMR (500 MHz, CDCl3): δ 7.46–7.25 (m, 10 H, Ar-H), 5.49 (s, 1 H, PhCH),
5.30–5.28 (m, 1 H, H-2B), 5.24 (dd, J = 9.9 and 3.5 Hz, 1 H, H-3B), 5.09 (br s,
1 H, H-1B), 4.90 (t, J = 9.9 Hz, 1 H, H-4B), 4.71 (d, J = 12 Hz, 1 H, PhCH2),
4.56 (d, J = 12.1 Hz, 1 H, PhCH2), 4.51 (d, J = 3.4 Hz, 1 H, H-1A), 4.25–4.20
(m, 1 H, H-6aA), 4.16–4.07 (m, 2 H, H-4A and H-5B), 3.79–3.76 (m, 1 H, H-5A),
3.69–3.46 (m, 1 H, H-6bA), 3.53–3.46 (m, 2 H, H-2A and H-3A), 3.35 (s, 3 H,
OCH3), 2.09 (s, 3 H, COCH3), 1.98 (s, 3 H, COCH3), 1.94 (s, 3 H, COCH3),

R. Panchadhayee & A.K. Misra
44
0.75 (d, J = 6.1 Hz, 3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 169.9 (COCH3),
169.7 (COCH3), 169.5 (COCH3), 137.6–126.3 (Ar-C), 101.7 (PhCH), 98.6 (C-1A),
97.9 (C-1B), 80.4 (C-2A), 79.7, (PhCH2), 74.1 (C-5A), 73.3, (C-3B), 71.1, (C-3A),
69.7 (C-4A), 69.3 (C-2B), 68.9 (C-4B), 65.9, (C-5B), 62.2 (C-6A), 55.3 (OCH3), 20.8
(COCH3), 20.7 (COCH3), 20.6 (COCH3), 16.6 (CCH3); ESI-MS: 667.2 [M+Na]+;
Anal. Calcd. for C33H40O13 (644.25): C, 61.48; H, 6.25; found: C, 61.30;
H, 6.50.
Methyl (2,3,4-tri-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-2-O-
benzyl-4,6-O-benzylidene-α-D-glucopyranoside (7)
To a solution of compound 6 (2 g, 3.10 mmol) in THF (15 mL) were added
powdered NaOH (1 g, 25 mmol), benzyl bromide (1.7 mL, 14.3 mmol), and
Bu4NBr (200 mg, 0.62 mmol) and the reaction mixture was allowed to stir
briskly at rt for 6 h. The reaction mixture was poured into H2O (300 mL) and
extracted with CH2Cl2 (150 mL). The organic layer was washed with H2O (200
mL), dried (Na2SO4), and concentrated under reduced pressure. The crude
product was purified over SiO2 using hexane-EtOAc (8:1) to give pure 7 (2.2
g, 90%). Yellow oil; IR (neat): 3031, 2979, 2899, 2867, 1497, 1454, 1389, 1361,
1
1181, 1123, 1093, 1059, 1046, 1028, 995, 912, 750 cm−1; H NMR (500 MHz,
CDCl3): δ 7.46–7.16 (m, 25 H, Ar-H), 5.45 (s, 1 H, PhCH), 5.17 (br s, 1 H, H-1B),
4.86 (d, J = 11.1 Hz, 1 H, PhCH2), 4.61–4.39 (m, 7 H, 3 PHCH2 and H-1A),
4.24–4.19 (m, 1 H, H-6aA), 4.12 (t, J = 9.3 Hz, H-4A), 4.01–3.97 (m, 1 H, H-5B),
3.84–3.74 (m, 3 H, H-2B, H-5A, and H-6bA), 3.65 (t, J = 4.9 Hz, 1 H, H-4B),
3.53 (t, J = 9.5 Hz, 1 H, H-3A), 3.41–3.36 (m, 2 H, H-2A and H-3B), 3.35 (s,
3 H, OCH3), 0.90 (d, J = 6.1 Hz, 3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ
139.0–126.3 (Ar-C), 101.6 (PhCH), 98.6 (C-1A), 98.4 (C-1B), 80.7 (C-2A), 80.4 (C-
5A), 80.1 (2 C, 2 PhCH2), 74.9 (2 C, 2 PhCH2), 74.4 (C-3B), 72.8 (C-3A), 72.1 (C-
2B), 72.0 (C-4A), 68.9 (C-4B), 67.7 (C-5B), 62.6 (C-6A), 55.5 (OCH3), 17.4 (CCH3);
ESI-MS: 811.3 [M+Na]+; Anal. Calcd. for C48H52O10 (788.36): C, 73.08; H, 6.64;
found: C, 72.86; H, 6.87.
Methyl (2,3,4-tri-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-2-O-
benzyl-α-D-glucopyranoside (8)
To a solution of compound 7 (2 g, 2.53 mmol) in CH3CN-H2O (50 mL, 9:1
v/v) was added HClO4-SiO2 (500 mg) and the reaction mixture was allowed to
stir at rt for 20 min. The reaction mixture was filtered and concentrated un-
der reduced pressure. The crude product was purified over SiO2 using hexane-
EtOAc (3:1) as eluant to give pure 8 (1.4 g, 79%). Yellow oil; IR (neat): 3364,
2922, 2857, 1497, 1454, 1370, 1207, 1121, 1060, 1027, 735 cm−1; 1H NMR (500
MHz, CDCl3): δ 7.33–7.23 (m, 20 H, Ar-H), 5.04 (d, J = 1.4 Hz, 1 H, H-1B), 4.94
(d, J = 10.8 Hz, 1 H, PhCH2) 4.68 (d, J = 12.3 Hz, 1 H, PhCH2), 4.64 (d, J =

Synthesis of Escherichia coli O158
45
10.8 Hz, 1 H, PhCH2), 4.63 (d, J = 12.3 Hz, 1 H, PhCH2) 4.59–4.53 (m, 3 H,
PhCH2), 4.51 (d, J = 3.6 Hz, 1 H, H-1A), 4.39 (d, J = 12.1 Hz, 1 H, PhCH2),
3.95–3.92 (m, 1 H, H-5B), 3.86–3.64 (m, 2 H, H-2B and H-4B), 3.81–3.78 (m, 2
H, H-6aA and H-3B), 3.76–3.73 (m, 1 H, H-6bA), 3.68 (t, J = 9.3 Hz, 1 H, H-
4A), 3.59–3.55 (m, 1 H, H-5A), 3.40 (t, J = 9.1 Hz, 1 H, H-3A), 3.35 (s, 3 H,
OCH3), 3.30 (dd, J = 9.5 and 3.6 Hz, 1 H, H-2A), 1.34 (d, J = 6.1 Hz, 3 H,
CCH3); 13C NMR (125 MHz, CDCl3): δ 138.8–127.9 (Ar-C), 100.7 (C-1A), 98.4
(C-1B) 85.4 (C-2B), 80.5 (C-5A), 79.6 (C-3B), 78.0 (C-3A), 75.8 (PhCH2), 75.6 (C-
2A), 73.9 (PhCH2), 73.0 (PhCH2), 72.4 (PhCH2), 71.1 (C-4A), 70.8 (C-4B), 69.8
(C-5B), 62.9 (C-6A), 55.6 (OCH3), 18.4 (CCH3); ESI-MS: 723.3 [M+Na]+; Anal.
Calcd. for C41H48O10 (700.32): C, 70.27; H, 6.90; found: C, 70.04; H, 7.15.
Methyl (2,3,4-tri-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-[2,3,4,6-
tetra-O-benzyl-α-D-glucopyranosyl)-(1→6)]-2-O-benzyl-α-D-
glucopyranoside (9)
To a solution of compound 8 (1 g, 1.43 mmol) and compound 5 (1 g, 1.71
˚
mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4A (1 g) and the reaction
mixture was allowed to stir at rt for 1 h then cooled to −40◦C. To the cold
reaction mixture were added NIS (450 mg, 2 mmol) and TMSOTf (10 µL) and
the reaction mixture was allowed to stir at the same temperature for 1 h. The
reaction mixture was filtered through a Celite bed and washed with CH2Cl2
(100 mL). The organic layer was successively washed with 5% Na2S2O3, satd.
NaHCO3, and H2O; dried (Na2SO4); and concentrated under reduced pressure.
The crude product was purified over SiO2 using hexane-EtOAc (4:1) as eluant
to give pure 9 (1.4 g, 80%). Yellow oil; IR (neat): 3064, 3030, 2919, 2870, 1730,
1496, 1454, 1362, 1216, 1061, 1028, 912, 753 cm−1; 1H NMR (500 MHz, CDCl3):
δ 7.31–7.11 (m, 40 H, Ar-H), 5.03 (d, J = 1.4 Hz, 1 H, H-1B), 5.0–4.81 (m, 3 H,
PhCH2), 4.83 (d, J = 3.8 Hz, 1 H, H-1C), 4.88–4.76 (m, 3 H, PhCH2), 4.68–4.57
(m, 4 H, PhCH2), 4.56–4.50 (m, 6 H, H-1A and PhCH2), 4.49–4.47 (m, 1 H,
PhCH2), 4.44–4.33 (m, 1 H, PhCH2), 4.06–4.03 (m, 1 H, H-6aC), 3.98–3.88 (m,
1 H, H-5B), 3.86–3.76 (m, 4 H, H-2B, H-4B, H-6bC and H-5C), 3.75–3.73 (m, 1
H, H-6aA), 3.72–3.63 (m, 3 H, H-6bA, H-3B and H-4C), 3.62–3.59 (m, 2 H, H-4A
and H-5A), 3.58–3.56 (m, 1 H, H-3C), 3.55–3.43 (m, 2 H, H-3A and H-2C), 3.30
(s, 3 H, OCH3), 3.26 (dd, J = 9.5 and 3.6 Hz, 1 H, H-2A), 1.3 (d, J = 6.1 Hz,
3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 139.0–127.8 (Ar-C), 104.4 (C-1A),
100.8 (C-1B), 98.4 (C-1C), 85.1 (C-2B), 82.7 (C-5A), 80.5 (C-3A), 79.7 (C-3B), 78.2
(C-2A), 78.1 (C-5C), 76.1 (PhCH2), 75.8 (PhCH2), 75.7 (C-4C), 75.4 (C-3C), 75.3
(PhCH2), 75.0 (PhCH2), 73.9 (PhCH2), 73.8 (2 PhCH2), 73.1 (C-2C), 73.0 (C-
4A), 72.4 (PhCH2 and C-4B), 70.8 (C-6A), 70.5 (C-6C), 70.1 (C-5B), 55.6 (OCH3),
18.4 (CCH3); ESI-MS: 1245.5 [M+Na]+; Anal. Calcd. for C75H82O15 (1222.57):
C, 73.63; H, 6.76; found: C, 73.44; H, 7.0.

R. Panchadhayee & A.K. Misra
46
Methyl (α-L-rhamnopyranosyl)-(1→3)-[α-D-glucopyranosyl)-
(1→6)]-α-D-glucopyranoside (1)
To a solution of compound 9 (1 g, 0.82 mmol) in CH3OH (10 mL) was added
20% Pd(OH)2-C (200 mg) and the reaction mixture was allowed to stir at rt
under a positive pressure of hydrogen for 24 h. The reaction mixture was fil-
tered through a Celite bed and concentrated under reduced pressure to give
compound 1, which was passed through a column of LH-20 Sephadex gel us-
ing CH3OH-H2O (8:1) as eluant to give pure 1 (280 mg, 68%). White powder;
1
IR (KBr): H NMR (500 MHz, CD3OD): δ 4.97 (br s, 1 H, H-1B), 4.69 (br s, 1
H, H-1C), 4.39 (d, J = 3.4 Hz, 1 H, H-1A), 4.08–4.06 (m, 1 H, H-3B), 3.96–3.91
(m, 2 H, H-4B and H-2B), 3.84–3.80 (m, 1 H, H-6aC), 3.77–3.73 (m, 1 H, H-
5B), 3.72–3.60 (m, 4 H, H-6abA, H-4A and H-4C), 3.59–3.56 (m, 1 H, H-6bC),
3.50–3.46 (m, 2 H, H-5A and H-5C), 3.42–3.36 (m, 2 H, H-2A and H-2C), 3.36 (s,
1 H, OCH3), 3.34–3.28 (m, 1 H, H-3C), 3.24 (t, J = 9.2 Hz, 1 H, H-3C), 1.15 (d, J
= 6.2 Hz, 3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 103.1 (C-1A), 101.5 (C-1B),
99.8 (C-1C), 80.3 (C-4A), 76.2 (C-2A), 73.5 (C-3A), 72.3 (C-2C), 72.0 (C-5B), 71.0
(C-4C), 70.7 (C-2B and C-4B), 70.6 (C-5C), 70.5 (C-3C), 70.0 (C-5A), 69.1 (C-3B),
60.9 (C-6A), 60.8 (C-6C), 55.6 (OCH3), 16.9 (CCH3); ESI-MS: 525.1 [M+Na]+;
Anal. Calcd. for C19H34O15 (502.19): C, 45.42; H, 6.82; found: C, 45.20;
H, 7.10.
2-(4-Methoxyphenoxy) ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-
β-D-galactopyranoside (13)
To a solution of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosyl
trichloroacetimidate (11; 1 g, 2.1 mmol) in anhydrous CH3CN (10 mL) was
added 2-(4-methoxyphenoxy) ethanol (12; 550 mg, 3.27 mmol) and the solu-
tion was cooled to −20◦C. To the cold reaction mixture was added TMSOTf
(20 µL) and the reaction mixture was allowed to stir at the same temperature
for 1 h. The reaction was quenched by addition of Et3N (0.1 mL) and diluted
with CH2Cl2 (100 mL). The organic layer was successively washed with satd.
NaHCO3 and H2O, dried (Na2SO4), and concentrated to dryness. The crude
product was purified over SiO2 using hexane-EtOAc (4:1) as eluant to give
pure 13 (790 mg, 78%). Yellow oil; IR (neat): 3020, 2837, 2116, 1794, 1508,
1
1370, 1229, 1045, 827, 758 cm−1; H NMR (500 MHz, CDCl3): δ 6.88–6.81 (m,
4 H, Ar-H), 5.33 (d, J = 3.2 Hz, 1 H, H-4), 4.79 (dd, J = 10.3 and 3.3 Hz, 1
H, H-3), 4.52 (d, J = 8.1 Hz, 1 H, H-1), 4.20–4.09 (m, 5 H, H-6ab, OCH2ab
,
and OCH2a), 4.01–3.96 (m, 1 H, OCH2b), 3.88–3.85 (m, 1 H, H-5), 3.76 (s, 3 H,
OCH3), 3.71 (dd, J = 10.9 Hz, 1 H, H-2), 2.16 (s, 3 H, COCH3), 2.09 (s, 3 H,
COCH3), 2.03 (s, 3 H, COCH3); 13C NMR (125 MHz, CDCl3): δ 170.7 (COCH3),
170.4 (COCH3), 170.2 (COCH3), 103.1 (C-1), 71.4 (C-4), 71.1 (C-3), 69.1 (C-5),
68.2 (OCH2), 66.7 (OCH2), 61.6 (C-2), 61.1 (C-6), 56.1 (OCH3), 21.0 (3 C, 3

Synthesis of Escherichia coli O158
47
COCH3); ESI-MS: 504.1 [M+Na]+; Anal. Calcd. for C21H27N3O10 (481.17): C,
52.39; H, 5.65; found: C, 52.20; H, 5.90.
2-(4-Methoxyphenoxy) ethyl 2-azido-4,6-O-benzylidene-2-deoxy-
β-D-galactopyranoside (14)
A solution of compound 13 (700 mg, 1.45 mmol) in 0.1 M CH3ONa (10 mL)
was allowed to stir at rt for 3 h and neutralized with Amberlite IR 120 (H+)
resin. The reaction mixture was filtered and concentrated under reduced pres-
sure. To a solution of the deacetylated product in anhydrous CH3CN (5 mL)
was added benzaldehyde dimethylacetal (330 µL, 2.2 mmol) and p-TsOH (50
mg) and the reaction mixture was allowed to stir at rt for 12 h. The reaction
was quenched with Et3N (0.1 mL) and the solvents were removed under re-
duced pressure. The crude product was purified over SiO2 using toluene-EtOAc
1
(2:1) as eluant to give pure 14 (560 mg, 87%). Yellow oil; H NMR (500 MHz,
CDCl3): δ 7.51–7.49 (m, 2 H, Ar-H), 7.37–7.35 (m, 3 H, Ar-H), 6.87 (d, J = 9.1
Hz, 2 H, Ar-H), 6.81 (d, J = 9.1 Hz, 2 H, Ar-H), 5.53 (s, 1 H, PhCH), 4.42 (d, J
= 7.9 Hz, 1 H, H-1), 4.27 (dd, J = 12.5 and 1.3 Hz, 1 H, H-6A), 4.23–4.19 (m,
1 H, OCH2a), 4.15–4.12 (m, 3 H, OCH2ab and H-4), 4.01 (dd, J = 12.5 and 1.7
Hz, 1 H, H-6b), 3.97–3.93 (m, 1 H, OCH2b), 3.74 (s, 3 H, OCH3), 3.65 (dd, J =
10.8 Hz, 1 H, H-2), 3.53 (dd, J = 9.9 and 2.9 Hz, 1 H, H-3), 3.40–3.38 (m, 1 H,
H-5); 13C NMR (125 MHz, CDCl3): δ 154.4–115.0 (Ar-C), 102.8 (PhCH), 101.7
(C-1), 75.0 (C-5), 71.7 (C-4), 69.3 (C-3), 68.7 (OCH2), 68.4 (OCH2), 66.9 (C-6),
64.2 (C-2), 56.1 (OCH3); ESI-MS: 466.1 [M+Na]+; Anal. Calcd. for C22H25N3O7
(443.17): C, 59.59; H, 5.68; found: C, 59.41; H, 5.90.
2-(4-Methoxyphenoxy) ethyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-
α-D-galactopyranosyl)-(1→3)-2-azido-4,6-O-benzylidene-2-
deoxy-β-D-galactopyranoside (15)
A solution of compound 14 (500 mg, 1.13 mmol) and compound 11 (650
g, 1.37 mmol) in anhydrous CH2Cl2 (8 mL) was cooled to −20◦C. To the cold
reaction mixture was added TMSOTf (10 µL) and the reaction mixture was
allowed to stir at the same temperature for 1 h. The reaction was quenched
with Et3N (0.1 mL) and diluted with CH2Cl2 (50 mL). The organic layer was
washed with H2O, dried (Na2SO4), and concentrated. The crude product was
purified over SiO2 using hexane-EtOAc (5:1) as eluant to give pure 15 (640
mg, 75%). Yellow oil; IR (neat): 3396, 2926, 1646, 1509, 1373, 1233, 1057, 823
cm−1; 1H NMR (500 MHz, CDCl3): δ 7.55–7.53 (m, 2 H, Ar-H), 7.35–7.33 (m, 3
H, Ar-H), 6.85 (d, J = 9.2 Hz, 2 H, Ar-H), 6.82 (d, J = 9.2 Hz, 2 H, Ar-H), 5.58
(s, 1 H, PhCH), 5.51 (d, J = 2.4 Hz, 1 H, H-4B), 5.43 (dd, J = 11.3 and 3.3 Hz,
1 H, H-3B), 5.21 (d, J = 3.6 Hz, 1 H, H-1B), 4.53 (t, J = 6.5 Hz, 1 H, H-5B), 4.49
(d, J = 8.0 Hz, 1 H, H-1A), 4.31 (dd, J = 12.5 and 1.2 Hz, 1 H, H-6aA), 4.27 (d,

R. Panchadhayee & A.K. Misra
48
J = 3.4 Hz, 1 H, H-4A), 4.25–4.22 (m, 1 H, OCH2a), 4.17–4.13 (m, 3 H, H-6abB
and OCH2a), 4.09 (dd, J = 12.4 and 1.4 Hz, 1 H, H-6bA), 4.06–4.01 (m, 1 H,
OCH2b), 4.00–3.96 (m, 1 H, OCH2b), 3.90 (dd, J = 8.1 Hz, 1 H, H-2A), 3.7 (s, 3
H, OCH3), 3.66 (dd, J = 11.2 and 3.3 Hz, 1 H, H-2B), 3.61 (dd, J = 10.5 and
3.6 Hz, 1 H, H-3A), 3.42–3.39 (m, 1 H, H-5A), 2.13, 2.03 (2 s, 9 H, 3 COCH3);
13C NMR (125 MHz, CDCl3): δ 170.8 (COCH3), 170.3 (COCH3), 169.9 (COCH3),
129.3–115.0 (Ar-C), 103.1 (C-1A), 101.3 (PhCH), 95.1 (C-1B), 75.0 (C-3A), 71.3
(C-4A), 69.4 (C-6A), 68.5 (C-6B), 68.4 (C-4B), 68.1 (C-3B), 68.0 (C-5B), 67.7 (C-
5A), 66.8 (OCH2), 62.0 (OCH2), 61.4 (C-2A), 57.3 (C-2B), 56.1 (OCH3), 21.0 (3 C,
3 COCH3); ESI-MS: 779.2 [M+Na]+; Anal. Calcd. for C34H40N6O14 (756.26): C,
53.97; H, 5.33; found: C, 53.76; H, 5.55.
2-(4-Methoxyphenyl)-ethyl (2-acetamido-2-deoxy-α-D-
galactopyranosyl)-(1→3)-2-acetamido-2-deoxy-β-D-
galactopyranoside (2)
To a solution of compound 15 (600 mg, 0.79 mmol) in CH3OH (5 mL)
was added 20% Pd(OH)2-C (100 mg) and the reaction mixture was allowed
to stir under hydrogen at rt for 12 h. The reaction mixture was filtered
through a Celite bed and concentrated. A solution of the crude product in acetic
anhydride-pyridine (5 mL, 1:1 v/v) was kept at rt for 2 h and the solvents were
removed under reduced pressure. A solution of the acetylated product in 0.1
M CH3ONa in CH3OH (5 mL) was allowed to stir at rt for 6 h and neutralized
with Dowex 50W X8 (H+) resin. The reaction mixture was filtered and concen-
trated under reduced pressure to give 2, which was further purified through
a column of Sephadex LH-20 using CH3OH as eluant (325 mg, 72%). White
powder; IR (KBr): 2934, 2114, 1751, 1508, 1371, 1233, 1130, 1054, 823 cm−1
;
1H NMR (500 MHz, DMSO-d6): δ 6.87–6.81 (m, 4 H, Ar-H), 4.94 (d, J = 3.6
Hz, 1 H, H-1B), 4.56 (d, J = 8.0 Hz, 1 H, H-1A), 4.27 (d, J = 2.8 Hz, 1 H,
H-4A), 4.05–3.93 (m, 8 H, H-6abA, H-6abB, H-2B, H-3A and OCH2ab), 3.85–3.69
(m, 4 H, H-3B, H-4B and OCH2ab), 3.67 (s, 3 H, OCH3), 3.58–3.49 (m, 3 H, H-
5A, H-5B and H-2A), 1.77, 1.46 (2 s, 6 H, 2 NHCOCH3); 13C NMR (125 MHz,
DMSO-d6): δ 170.3 (2 NHCOCH3), 129.6–127.1 (Ar-C), 101.7 (C-1A), 94.6 (C-
1B), 71.9 (C-4A), 71.7 (C-4B), 69.4 (C-6A), 68.6 (C-6B), 68.4 (C-3B), 68.3 (2 C, 2
OCH2), 67.9 (C-3A and C-5B), 66.7 (C-5A), 60.9 (C-2A), 56.2 (OCH3), 50.5 (C-2B),
23.7 (NHCOCH3), 23.3 (NHCOCH3); ESI-MS: 597.2 [M+Na]+; Anal. Calcd. for
C25H38N2O13 (574.24): C, 52.26; H, 6.67; found: C, 52.02; H, 6.95.
ACKNOWLEDGEMENTS
R.P. thanks CSIR, New Delhi, for providing a Senior Research Fellowship. This
work was supported by Ramanna Fellowship (AKM), Department of Science
and Technology, New Delhi (SR/S1/RFPC-06/2006).

Synthesis of Escherichia coli O158
49
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