Concise synthesis of the pentasaccharide O-antigen corresponding to the Shiga toxin producing Escherichia coli O171

Article abstract of DOI:10.1016/j.bioorg.2010.01.001

An expedient total synthesis of a pentasaccharide as its 4-methoxyphenyl glycoside corresponding to the Shiga toxin producing Escherichia coli O171 has been achieved for the first time in excellent yield. Most of the glycosylation steps are highly stereoselective. Stereoselective glycosylation of sialic acid derivative was obtained exploiting the nitrile effect of the solvent used.

Full text of DOI:10.1016/j.bioorg.2010.01.001

Bioorganic Chemistry 38 (2010) 56–61
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Concise synthesis of the pentasaccharide O-antigen corresponding to the Shiga
toxin producing Escherichia coli O171
*
Pintu Kumar Mandal, Anup Kumar Misra
Division of Molecular Medicine, Bose Institute, A.J.C. Bose Birth Centenary Campus, P-1/12, C.I.T. Scheme VII-M, Kolkata 700 054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2009
Available online 11 January 2010
An expedient total synthesis of a pentasaccharide as its 4-methoxyphenyl glycoside corresponding to the
Shiga toxin producing Escherichia coli O171 has been achieved for the first time in excellent yield. Most of
the glycosylation steps are highly stereoselective. Stereoselective glycosylation of sialic acid derivative
was obtained exploiting the nitrile effect of the solvent used.
Keywords:
Ó 2010 Elsevier Inc. All rights reserved.
Oligosaccharides
Glycosylations
Sialic acid
Escherichia coli O171
Pentasaccharide
1. Introduction
ous intestinal infections and associated with several outbreaks of
disease in the developed countries [7,8]. Besides E. coli O157:H7,
Escherichia coli is a group of Gram-negative bacteria that colo-
nize in the infant’s gastrointestinal tract within hours of birth
[1]. Escherichia has been classified by a series of physiological
and morphological traits. Based on the immunogenicity of the sur-
face oligosaccharides [2], E. coli have been divided into a number of
serotypes. There are three types of E. coli antigens which include:
(i) somatic (O) antigen, (ii) capsular (K) antigen and (iii) flagellar
(H) antigen. Although E. coli is generally confined to the intestinal
lumen, in an immunosuppressed host ‘‘nonpathogenic” strains of
E. coli can also cause infections [3]. In general, three common infec-
tions, e.g. (i) enteric/diarrhoeal, (ii) urinary tract infections and (iii)
septicaemia/meningitis caused by virulent E. coli strains [4]. Path-
ogenic E. coli strains are divided in six classes, which are: (i) enter-
opathogenic E. coli (EPEC), (ii) enteroinvasive E. coli (EIEC), (iii)
enterotoxigenic E. coli (ETEC), (iv) enteroaggregative E. coli (EAEC),
(v) diffusely adherent E. coli (DAEC), and (vi) enterohemorrhagic
E. coli (EHEC) [5].
Enterohemorrhagic E. coli (EHEC) strains act as the etiological
agent of diarrhoea with lifethreatening complications like haemor-
rhagic colitis (HC) and haemolytic–uraemic syndrome (HUS). EHEC
strains show considerable toxic effect on the cultured Vero cells
and also known as ‘‘verotoxigenic E. coli” (VTEC) strains. They are
also termed as ‘‘Shiga toxin producing E. coli” (STEC) because of
their ability to produce bacteriophage-mediated Shiga-like toxin
[6]. The well documented Shiga toxin producing EHEC strain is
E. coli O157:H7, which is responsible for the frequent cause of seri-
several other E. coli serotypes have been reported to be associated
with the STEC class [9]. Recent structural analysis of the O-antigen
of Shiga toxin producing E. coli O171 showed that it contains an
acidic pentasaccharide repeating unit having an N-acetylneurami-
nic acid (sialic acid) at the non-reducing terminus [10]. Several
studies in the past have established that the oligosaccharide epi-
topes of the bacterial O-antigens have strong influence on the
immunochemical activities of the glycoconjugate vaccines derived
from them. Therefore, bacterial O-antigens have been chosen for
the development of glycoconjugate vaccine candidates against
infectious diseases [11]. Because of the limited availability of the
desired cell-wall pentasaccharide of E. coli O171 from the natural
source, it is highly essential to develop a concise chemical syn-
thetic strategy for the preparation of the target pentasaccharide
for its ready availability in the biological studies. In the immuno-
chemical studies, most often it is required to conjugate the oligo-
saccharide moiety with a carrier protein through a spacer linker.
Therefore, synthesis of the target pentasaccharide with a tempo-
rary protecting group at the reducing end would be beneficial for
its ready removal after construction of the pentasaccharide. As a
part of our ongoing program on the synthesis of complex oligosac-
charides corresponding to the microbial cell-wall polysaccharides
[12], we describe herein an efficient chemical synthesis of the sialic
acid containing pentasaccharide repeating unit of the O-antigen of
Shiga toxin producing E. coli O171 as its 4-methoxyphenyl glyco-
side involving a block synthetic strategy (Fig. 1). 4-Methoxyphenyl
group has been chosen as the temporary anomeric protecting
group because of its easy removal after construction of the
pentasaccharide.
* Corresponding author. Fax: +91 33 2355 3886.
E-mail address: akmisra69@gmail.com (A.K. Misra).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.01.001

P.K. Mandal, A.K. Misra / Bioorganic Chemistry 38 (2010) 56–61
57
2.3. 4-Methoxyphenyl (4,6-O-benzylidene-b-D-galactopyranosyl)-
HO
OH
COONa
(1 ? 3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-
D-
HO
E
O
galactopyranoside (8)
AcHN
O
HO
HO
O
D
A solution of compound 7 (2.5 g, 2.98 mmol) in 0.1 M CH3ONa
in CH3OH (100 mL) was allowed to stir at room temperature for
30 min and neutralized with Amberlite-IR 120 (H+) resin. The reac-
tion mixture was filtered and evaporated to dryness to give the
crude product, which was passed through a short column of SiO2
using hexane–EtOAc (2:1) as eluant to give pure 8 (2.2 g, quantita-
HO
O
HO OH
HO OH
HO
HO
HO
O
O
O
A
B
OMp
NHAc
C
O
O
HO
Mp:4-methoxyphenyl
HO
1
tive): Rf (0.2, toluene–EtOAc: 2:1); Yellow oil; ½a D25
ꢁ
+27.5 (c 1.5,
Fig. 1. Chemical structure of the synthesized pentasaccharide repeating unit of
E. coli O171 as its 4-methoxyphenyl glycoside.
CHCl3); IR (neat): 3502, 2926, 1751, 1716, 1508, 1388, 1369,
1222, 1080, 1043, 952, 699 cmꢀ1 1H NMR (300 MHz, CDCl3): d
;
7.85–7.23 (m, 14H, Ar-H), 6.88 (d, J = 9.1 Hz, 2H, Ar-H), 6.69 (d,
J = 9.1 Hz, 2H, Ar-H), 5.69 (d, J = 8.3 Hz, 1H, H-1A), 5.51 (s, 1H,
PhCH), 5.19 (s, 1H, PhCH), 4.75 (t, J = 8.3 Hz, 1H, H-2A), 4.73 (dd,
J = 10.8, 3.2 Hz, 1H, H-3A), 4.59 (d, J = 8.0 Hz, 1H, H-1B), 4.34 (brs,
1H, H-4A), 4.23 (d, J = 12.0 Hz, 1H, H-6aA), 3.95 (d, J = 11.8 Hz, 1H,
H-6bA), 3.77 (brs, 1H, H-4B), 3.74–3.71 (m, 1H, H-6aB), 3.67 (s, 3H,
OCH3), 3.56–3.47 (m, 2H, H-6bB, H-3B), 3.17–3.16 (m, 1H, H-5B),
3.12–2.82 (m, 1H, H-2B), 2.40–2.36 (m, 1H, H-5A); 13C NMR
(75 MHz, CDCl3): d 168.3, 167.4 (COPhth), 155.5–114.3 (Ar-C),
101.0 (PhCH), 100.7 (PhCH), 99.3 (C-1B), 97.8 (C-1A), 75.5 (C-4B),
74.3 (C-4A), 72.5 (C-3A), 69.2 (C-2B), 68.9 (2C, C-3B, C-6B), 68.1 (C-
6A), 66.6 (C-5A), 63.8 (C-5B), 55.6 (OCH3), 52.2 (C-2A); ESI-MS: m/
z 776.8 [M+Na]+; Anal. Calcd for C41H39NO13 (753.24): C, 65.33;
H, 5.22. Found: C, 65.10; H, 5.46.
2. Experimental
2.1. General
All reactions were monitored by thin layer chromatography
over silica gel coated TLC plates. The spots on TLC were visualized
by warming ceric sulphate (2% Ce(SO4)2 in 2 N H2SO4) sprayed
plates on a hot plate. Silica gel 230–400 mesh was used for column
chromatography. 1H and 13C NMR, 2D COSY, HMQC spectra were
recorded on Brucker Avance DRX 500 MHz using CDCl3 and D2O
as solvents and TMS as internal reference unless stated otherwise.
Chemical shift value is expressed in d ppm. ESI-MS were recorded
on a Micromass Quttro II triple quadrupole mass spectrometer. Ele-
mentary analysis was carried out on Carlo ERBA-1108 analyzer.
Optical rotations were measured at 25 °C on a Perkin Elmer 341
polarimeter. Commercially available grades of organic solvents of
adequate purity are used in many reactions.
2.4. Phenyl (2,3,4-tri-O-acetyl-6-O-benzyl-b-D-galactopyranosyl-
(1 ? 6)-2,3,4-tri-O-acetyl-1-thio-b- -glucopyranoside (10)
D
To a solution of compound 4 (2.5 g, 5.67 mmol) in CH3CN–H2O
(30 mL; 20:1 v/v) was added N-iodosaccharin (3.5 g, 11.32 mmol)
and the reaction mixture was allowed to stir for 15 min at room
temperature. The reaction mixture was diluted with CH2Cl2
(100 mL) and successively washed with 5% aq. Na2S2O3 and water.
The organic layer was dried (Na2SO4) and concentrated under re-
duced pressure. The crude product was purified over SiO2 using
hexane–EtOAc (5:1) as eluant to give pure hemiacetal derivative,
which was used immediately for the next step. To a solution of
the hemiacetal derivative (1.9 g, 4.79 mmol) in anhydrous CH2Cl2
(25 mL) was added CCl3CN (3 mL, 29.91 mmol) and the reaction
mixture was cooled to ꢀ10 °C. To the cold reaction mixture was
2.2. 4-Methoxyphenyl (2,3-di-O-acetyl-4,6-O-benzylidene-b-
galactopyranosyl)-(1 ? 3)-4,6-O-benzylidene-2-deoxy-2-N-
D-
phthalimido-b- -galactopyranoside (7)
D
To a solution of compound 2 (2 g, 3.97 mmol) and thioglycoside
donor 3 (2 g, 5.04 mmol) in CH2Cl2 (25 mL) was added MS-4 Å (5 g)
and the reaction mixture was allowed to stir at room temperature
under argon for 30 min. The reaction mixture was cooled to ꢀ25 °C
and N-iodosuccinimide (1.3 g, 5.77 mmol) and TMSOTf (20 lL)
were added to it. After stirring the reaction mixture at the same
temperature for 1 h, it was filtered through a CeliteÒ bed and
washed with CH2Cl2 (100 mL). The organic layer was washed with
5% Na2S2O3 (100 mL), satd. NaHCO3 (100 mL) and water (100 mL)
in succession, dried (Na2SO4) and evaporated to dryness. The crude
mass was purified over SiO2 using hexane–EtOAc (5:1) as eluant to
furnish pure 7 (2.8 g, 84%): Rf (0.4, toluene–EtOAc: 4:1); colorless
added DBU (100 lL, 0.67 mmol) and the reaction mixture was stir-
red at 5 °C for 6 h and evaporated to dryness under reduced pres-
sure. The crude product was purified over SiO2 using hexane–
EtOAc (6:1) as eluant to give pure compound 9, which was used
immediately for the glycosylation. To a solution of compound 9
(2.3 g, 4.25 mmol) in anhydrous CH2Cl2 (25 mL) were added com-
pound 5 (1.4 g, 3.51 mmol) and MS-4 Å (3 g) and the reaction mix-
ture was allowed to cool at ꢀ30 °C. To the cold reaction mixture
oil; ½a 2D5
ꢁ
+41.3 (c 1.5, CHCl3); IR (neat): 3443, 2841, 1714, 1510,
1390, 1269, 1089, 999, 704 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d
7.89–7.23 (m, 14H, Ar-H), 6.81 (d, J = 9.0 Hz, 2H, Ar-H), 6.64 (d,
J = 9.0 Hz, 2H, Ar-H), 5.64 (d, J = 8.5 Hz, 1H, H-1A), 5.47 (s, 1H,
PhCH), 5.19 (s, 1H, PhCH), 4.98 (dd, J = 10.8, 3.6 Hz, 1H, H-3B),
4.88–4.83 (m, 2H, H-2B, H-2A), 4.69 (dd, J = 11.2, 3.3 Hz, 1H, H-
3A), 4.60 (d, J = 8.0 Hz, 1H, H-1B), 4.32–4.25 (m, 2H, H-4B, H-6aB),
4.06–3.99 (m, 2H, H-4A, H-6bB), 3.71–3.68 (m, 1H, H-6aA), 3.64 (s,
3H, OCH3), 3.57 (brs, 1H, H-5A), 3.47–3.43 (m, 1H, H-6bA), 3.17
(brs, 1H, H-5B), 1.92, 1.50 (2s, 6H, 2COCH3); 13C NMR (75 MHz,
CDCl3): d 170.5, 170.2 (2COCH3), 168.7, 167.3 (COPhth), 155.4–
114.5 (Ar-C), 101.1 (C-1B), 100.9 (PhCH), 100.6 (PhCH), 97.9 (C-
1A), 74.9 (C-4B), 73.3 (C-4A), 72.5 (C-3A), 69.0 (C-6B), 68.8 (C-6A),
67.8 (2C, C-2B, C-3B), 66.8 (C-5A), 62.6 (C-5B), 55.5 (OCH3), 54.6
(C-2A), 20.8, 20.2 (2COCH3); ESI-MS: m/z 860.8 [M+Na]+; Anal.
Calcd for C45H43NO15 (837.26): C, 64.51; H, 5.17. Found: C, 64.32;
H, 5.43.
was added TMSOTf (30 lL) and the reaction mixture was allowed
to stir at ꢀ20 °C for 1 h. The reaction was quenched with Et3N
(0.1 mL), filtered and washed with CH2Cl2 (100 mL). The organic
layer was washed with satd. aq. 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 10 (2.1 g, 77%): Rf (0.3, hexane–EtOAc: 2:1); colorless
oil; ½a 2D5
ꢁ
ꢀ29.8 (c 1.5, CHCl3); IR (neat): 2953, 1761, 1712, 1379,
1382, 1223, 1069, 699 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d 7.39–
7.26 (m, 10H, Ar-H), 5.45 (d, J = 2.9 Hz, 1H, H-4D), 5.19 (t, J =
9.4 Hz, 1H, H-2D), 5.17 (dd, J = 10.4, 8.0 Hz, 1H, H-3C), 5.00 (dd,
J = 10.0, 3.4 Hz, 1H, H-3D), 4.98 (t, J = 9.7 Hz, 1H, H-2C), 4.89
(t, J = 9.6 Hz, 1H, H-4C), 4.55 (d, J = 11.9 Hz, 1H, PhCH2), 4.51 (d,
J = 7.9 Hz, 1H, H-1D), 4.46 (d, J = 10.0 Hz, H-1C), 4.42 (d,
J = 11.9 Hz, 1H, PhCH2), 3.89 (dd, J = 11.0, 2.2 Hz, 1H, H-6aC),

58
P.K. Mandal, A.K. Misra / Bioorganic Chemistry 38 (2010) 56–61
3.84–3.83 (m, 1H, H-5D), 3.70–3.67 (m, 1H, H-5C), 3.60–3.53 (m,
2H, H-6bC, H-6aD), 3.48 (dd, J = 10.0, 6.6 Hz, 1H, H-6bD), 2.07, 2.05,
2.04, 1.99, 1.98, 1.97 (6s, 18H, 6COCH3); 13C NMR (125 MHz,
CDCl3): d 170.5 (2C), 170.4, 170.0 169.9, 169.8 (6COCH3), 137.9–
128.3 (Ar-C), 101.7 (C-1D), 83.5 (C-1C), 77.7 (C-5C), 74.3 (C-2D),
73.9 (PhCH2), 72.6 (C-5D), 71.5 (C-3D), 70.3 (C-2C), 69.5 (C-3C),
69.3 (C-4C), 68.9 (C-6C), 67.9 (C-4D), 67.8 (C-6D), 21.2, 21.1, 21.0,
20.9, 20.8 (2C, 6COCH3); ESI-MS: m/z 799.3 [M+Na]+; Anal. Calcd
for C37H44O16S (776.24): C, 57.21; H, 5.71. Found: C, 57.02; H, 6.0.
with toluene (3 ꢂ 30 mL). To a solution of the crude product in pyr-
idine (10 mL) was added acetic anhydride (10 mL) and the reaction
mixture was stirred at room temperature for 3 h. The solvents were
removed under reduced pressure and co-evaporated with toluene
(3 ꢂ 30 mL). To a solution of the acetylated product in CH3OH
(30 mL) was added 20% Pd(OH)2–C (100 mg) and the reaction mix-
ture was allowed to stir under a positive pressure of hydrogen at
room temperature for 4 h. The reaction mixture was filtered
through a CeliteÒ bed and evaporated to dryness. The crude prod-
uct was passed through a short pad of SiO2 using hexane–EtOAc
(1:1) as eluant to give pure 12 (1.4 g, 64%); Rf (0.2, hexane–EtOAc:
2.5. 4-Methoxyphenyl (2,3,4-tri-O-acetyl-6-O-benzyl-b-
pyranosyl-(1 ? 6)-(2,3,4-tri-O-acetyl-b- -glucopyranosyl)-(1 ? 3)-
(2-O-acetyl-4,6-O-benzylidene-b- -galactopyranosyl)-(1 ? 3)-4,6-O-
benzylidene-2-deoxy-2-N-phthalimido-b- -galactopyranoside (11)
D-galacto-
D
1:2); colorless oil; ½a D25
ꢁ
+13.2 (c 1.5, CHCl3); IR (neat): 3481, 2924,
D
1753, 1714, 1508, 1371, 1246, 1222, 1047, 754 cmꢀ1 1H NMR
;
D
(500 MHz, CDCl3): d 7.80–7.27 (m, 10H, Ar-H), 6.87 (d, J = 9.1 Hz,
2H, Ar-H), 6.72 (d, J = 9.1 Hz, 2H, Ar-H), 5.71 (d, J = 8.4 Hz, 1H, H-
1A), 5.56 (s, 1H, PhCH), 5.35 (d, J = 3.4 Hz, 1H, H-4D), 5.31 (s, 1H,
PhCH), 5.20 (dd, J = 10.4, 3.3 Hz, 1H, H-3C), 5.08 (t, J = 9.4 Hz, 1H,
H-2D), 5.03–4.97 (m, 3H, H-2B, H-3D, H-4C), 4.91–4.87 (m, 2H, H-
2C, H-2A), 4.77 (dd, J = 10.8, 3.4 Hz, 1H, H-3A), 4.68 (d, J = 8.0 Hz,
1H, H-1B), 4.66 (d, J = 8.0 Hz, 1H, H-1D), 4.47 (d, J = 8.0 Hz, 1H, H-
1C), 4.34 (d, J = 12.3 Hz, 1H, H-6aC), 4.25 (d, J = 3.4 Hz, 1 H, H-4B),
4.12 (d, J = 11.2 Hz, 1H, H-6bC), 4.00–3.97 (m, 2H, H-4A, H-3B),
3.93–3.90 (m, 1H, H-6aD), 3.71 (s, 3H, OCH3), 3.69–3.56 (m, 4H,
H-6abB, H-6aA, H-6bD), 3.47–3.39 (m, 1H, H-6bA), 3.37–3.32 (m,
3H, H-5C, H-5D, H-5B), 3.15 (brs, 1 H, H-5A), 2.16, 2.15, 2.04, 2.02,
2.01, 1.96, 1.89 (7s, 21 H, 7COCH3), 1.78 (s, 3 H, NHCOCH3); 13C
NMR (125 MHz, CDCl3): d 171.2, 170.8, 170.7, 170.5, 170.1, 169.9,
169.3 (7COCH3), 168.9 (NHCOCH3), 156.0–114.8 (Ar-C), 101.9
(PhCH), 101.5 (C-1C), 101.4 (C-1D), 100.7 (PhCH), 98.5 (C-1A), 96.6
(C-1B), 75.9 (C-4A), 74.4 (C-3A), 74.2 (C-5D), 73.5 (C-3B), 73.4 (C-
2D), 73.2 (C-4B), 73.1 (C-2C), 71.6 (C-2B), 71.5 (C-3D), 70.5 (C-4C),
69.7 (C-3C), 69.6 (C-6C), 69.2 (C-5C), 68.6 (C-6B), 68.5 (C-6D), 68.4
(C-4D), 67.1 (C-5B), 63.8 (C-5A), 61.2 (H-6A), 55.9 (OCH3), 52.7 (C-
2A), 21.3, 21.2 (2C), 21.1 (2C), 21.0, 20.9 (7COCH3), 20.8
(NHCOCH3); ESI-MS: m/z 1301.2 [M+NH4]+; Anal. Calcd for
C61H73NO29 (1283.43): C, 57.05; H, 5.73. Found: C, 56.90; H, 5.94.
To a solution of the disaccharide acceptor 8 (1.7 g, 2.25 mmol)
and the disaccharide thioglycoside donor 10 (2 g, 2.57 mmol) in
CH2Cl2 (20 mL) was added MS-4 Å (2 g) and the reaction mixture
was allowed to stir at room temperature for 1 h. The reaction mix-
ture was cooled to ꢀ40 °C and NIS (650 mg, 2.88 mmol) and
TMSOTf (10 lL) were added in succession. The reaction mixture
was allowed to stir at same temperature for 30 min and diluted
with CH2Cl2 (100 mL). The reaction mixture was filtered through
a CeliteÒ bed and the organic layer was washed with 5% aq.
Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4) and evaporated
to dryness. The crude mass was acetylated using acetic anhydride
(5 mL) and pyridine (5 mL) at room temperature. The acetylated
crude mass was purified over SiO2 using hexane–EtOAc (3:1) as
eluant to furnish pure 11 (2.6 g, 79%): Rf (0.5, hexane–EtOAc:
1:1); colorless oil; ½a D25
ꢁ
+12.5 (c 1.5, CHCl3); IR (neat): 2933,
1755, 1716, 1508, 1388, 1369, 1247, 1220, 1051, 754, 699 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d 7.63–7.24 (m, 19H, Ar-H), 6.87 (d,
J = 9.1 Hz, 2H, Ar-H), 6.72 (m, J = 9.1 Hz, 2H, Ar-H), 5.72 (d,
J = 8.4 Hz, 1H, H-1A), 5.55 (s, 1H, PhCH), 5.47 (d, J = 3.3 Hz, 1H, H-
4D), 5.33 (s, 1H, PhCH), 5.20 (dd, J = 10.8, 3.4 Hz, 1H, H-3C), 5.07
(t, J = 9.4 Hz, 1H, H-2D), 5.02–4.99 (m, 2H, H-3D, H-2B), 4.94–4.91
(m, 2H, H-2C, H-2A), 4.87 (t, J = 8.1 Hz, 1H, H-4C), 4.81 (dd,
J = 10.8, 3.2 Hz, 1H, H-3A), 4.63 (d, J = 7.9 Hz, 1H, H-1B), 4.60 (d,
J = 8.0 Hz, 1H, H-1D), 4.52 (d, J = 11.9 Hz, 1H, PhCH2), 4.46 (d,
J = 8.0 Hz, 1H, H-1C), 4.40 (d, J = 11.9 Hz, 1H, PhCH2), 4.34 (d,
J = 12.2 Hz, 1H, 6aC), 4.25 (d, J = 3.2 Hz, 1H, H-4B), 4.11 (d,
J = 11.0 Hz, 1H, 6bC), 4.02 (d, J = 3.2 Hz, 1H, H-4A), 3.94 (dd,
J = 10.9, 3.5 Hz, 1H, H-3B), 3.90 (d, J = 8.7 Hz, 1H, H-6aD), 3.84–
3.82 (m, 1H, H-5D), 3.76 (brs, 1H, H-5B), 3.71 (s, 3H, OCH3), 3.62–
3.44 (m, 6H, H-6bD, H-6abA, H-6abB, H-5C), 3.17 (brs, 1H, H-5A),
2.06, 2.03, 2.00, 1.97, 1.95, 1.87, 1.72 (7s, 21H, 7COCH3); 13C
NMR (125 MHz, CDCl3): d 170.7, 170.6, 170.5, 170.4, 169.9, 169.8,
169.3 (7COCH3), 168.9, 167.9 (COPhth), 155.9–114.8 (Ar-C), 101.9
(C-1C), 101.8 (PhCH), 101.5 (C-1D), 100.7 (PhCH), 98.5 (C-1A), 96.1
(C-1B), 75.8 (C-4A), 73.9 (2C, PhCH2, C-3A), 73.7 (C-3B), 73.4 (C-
2D), 73.2 (C-3D), 72.9 (C-4B), 72.6 (C-4C), 71.6 (2C, C-2B, C-4D),
70.5 (C-6C), 69.6 (C-6A), 69.4 (C-6B), 69.1 (C-2C), 68.6 (C-6D), 68.0
(C-3C), 67.9 (C-5D), 67.1 (C-5C), 63.7 (C-5A), 57.2 (C-5B), 55.9
(OCH3), 52.7 (C-2A), 21.2, 21.1 (2C), 21.0, 20.9 (2C), 20.8 (7COCH3);
ESI-MS: m/z 1484.5 [M+Na]+; Anal. Calcd for C74H79NO30
(1461.47): C, 60.78; H, 5.44. Found: C, 60.57; H, 5.67.
2.7. 4-Methoxyphenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
4-tri-O-acetyl-b-
glucopyranosyl)-(1 ? 3)-(2-O-acetyl-4,6-O-benzylidene-b-
D
-glycero-
-galactopyranosyl)-(1 ? 6)-(2,3,4-tri-O-acetyl-b-
-galacto-
a-D-galacto-2-nonulopyranosylonate)-(2 ? 6)-(2,3,-
D
D-
D
pyranosyl)-(1 ? 3)-2-acetamido-4,6-O-benzylidene-2-deoxy-b-
D-
galactopyranoside (13)
To a solution of compound 12 (1 g, 0.78 mmol) and thioglyco-
side donor 6 (800 mg, 1.37 mmol) in anhydrous CH3CN–CH2Cl2
(20 mL; 5:1 v/v) was added MS-3 Å (2 g) and the reaction mixture
was allowed to stir at room temperature under argon for 30 min.
The reaction mixture was cooled to ꢀ10 °C and N-iodosuccinimide
(350 mg, 1.55 mmol) and TfOH (5 lL) were added to it. After stir-
ring the reaction mixture at the same temperature for 20 h, it
was filtered through a CeliteÒ bed and washed with CH2Cl2
(100 mL). The organic layer was washed with 5% Na2S2O3
(100 mL), satd. NaHCO3 (100 mL) and water (100 mL) in succes-
sion, dried (Na2SO4) and evaporated to dryness. The crude mass
was purified over SiO2 using toluene–EtOAc (1:2) as eluant to fur-
nish pure 13 (725 mg, 53%); Rf (0.3, toluene–EtOAc: 1:4); colorless
2.6. 4-Methoxyphenyl (2,3,4-tri-O-acetyl-b-
(2,3,4-tri-O-acetyl-b- -glucopyranosyl)-(1 ? 3)-(2-O-acetyl-4,6-O-ben-
zylidene-b- -galactopyranosyl)-(1 ? 3)-2-acetamido-4,6-O-benzylide-
ne-2-deoxy-b- -galactopyranoside (12)
D-galactopyranosyl-(1 ? 6)-
oil; ½a 2D5
ꢁ
+19.5 (c 1.5, CHCl3); IR (neat): 3481, 2928, 1749, 1716,
D
1541, 1373, 1222, 1045, 756, 699 cmꢀ1; 1H NMR (500 MHz, CDCl3):
d 7.62–7.29 (m, 10H, Ar-H), 6.91 (d, J = 9.0 Hz, 2H, Ar-H), 6.69 (d,
J = 9.0 Hz, 2H, Ar-H), 5.73 (d, J = 8.5 Hz, 1H, H-1A), 5.56 (s, 1H,
PhCH), 5.49 (d, J = 3.4 Hz, 1H, H-4D), 5.35 (s, 1H, PhCH), 5.34–5.26
(m, 3H, H-7E, H-8E and NHCOCH3), 5.17 (dd, J = 10.1, 3.5 Hz, 1H,
H-3C), 5.09–5.05 (m, 2H, H-2D, H-2C), 5.03–4.97 (m, 2H, H-4E, H-
3D), 4.93–4.84 (m, 3H, H-2B, H-4C, H-2A), 4.78 (dd, J = 10.8, 3.4 Hz,
1H, H-3A), 4.67 (d, J = 8.0 Hz, 1H, H-1B), 4.58 (d, J = 8.0 Hz, 1H, H-
D
D
To a solution of compound 11 (2.5 g, 1.71 mmol) in n-butanol
(80 mL) was added ethylene diamine (0.5 mL, 7.47 mmol) and
the reaction mixture was allowed to stir at 100 °C for 6 h. The sol-
vents were removed under reduced pressure and co-evaporated

P.K. Mandal, A.K. Misra / Bioorganic Chemistry 38 (2010) 56–61
59
1D), 4.55 (d, J = 7.9 Hz, 1H, H-1C), 4.42–4.37 (m, 2H, H-9aE, H-6aC),
3. Results and discussion
4.37 (dd, J = 10.8, 3.5 Hz, 1H, H-3B), 4.29 (d, J = 3.4 Hz, 1H, H-4B),
4.16–4.09 (m, 2H, H-9bE, H-6bC), 4.06–4.01 (m, 4H, H-5E, H-6E, H-
4A, H-6aD), 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.95–3.68 (m,
6H, H-6abA, H-6abB, H-6bD, H-5C), 3.59–3.57 (m, 1H, H-5B), 3.18
(brs, 1H, H-5A), 2.71 (dd, J = 12.1, 3.8 Hz, 1H, H-3eE), 2.19, 2.16,
2.13, 2.06, 2.05, 2.04, 2.03, 2.02, 2.01, 1.97, 1.95, 1.89, 1.88 (13s,
39H, 13COCH3), 1.86–1.84 (m, 1H, H-3aE); 13C NMR (125 MHz,
CDCl3): d 171.3, 171.1, 170.9 (2C), 170.8 (2C), 170.7, 170.6, 170.5,
170.3, 170.2, 170.0 169.5 (13COCH3), 168.3 (COOCH3), 155.3–
114.5 (Ar-C), 101.9 (C-2E), 101.8 (PhCH), 101.7 (C-1C), 101.6 (C-
1D), 100.6 (PhCH), 99.5 (C-1A), 98.2 (C-1B), 75.7 (C-4A), 73.8 (C-
3A), 73.5 (C-2B), 73.2 (C-5D), 73.0 (2C, C-3B, C-2D), 72.3 (C-4B),
71.6 (C-5C), 71.5 (C-4C), 69.6 (C-2C), 69.3 (C-3C), 69.1 (C-3D), 69.0
(C-5E), 68.6 (C-7E), 68.3 (C-8E), 68.4 (2C, C-4E and C-6D), 68.2 (C-
6B), 67.9 (C-6A), 67.6 (2C, C-5B and C-4D), 67.1 (C-6C), 63.8 (C-9E),
63.0 (C-5A), 57.2 (COOCH3), 54.2 (C-6E), 53.7 (OCH3), 52.2 (C-2A),
38.3 (C-3E), 23.2 (2C), 21.4, 21.3 (2C), 21.2 (3C), 21.1, 21.0 (2C),
20.9, 20.8 (13COCH3); ESI-MS: m/z 1774.6 [M+NH4]+; Anal. Calcd
for C81H100N2O41 (1756.58): C, 55.35; H, 5.73. Found: C, 55.13; H,
5.97.
In order to synthesize the target pentasaccharide 1 as its 4-
methoxyphenyl glycoside, a block synthetic strategy has been
adopted in which a disaccharide thioglycoside (10) was stereose-
lectively coupled to a disaccharide acceptor (8) under iodonium
ion promoted thioglycoside activation condition. The tetrasaccha-
ride derivative (11) thus obtained was transformed to a tetrasac-
charide acceptor (12) for the final stereoselective glycosylation
with sialic acid derived thioglycoside donor towards the formation
of a sialylated tetrasaccharide derivative (13), which was deprotec-
ted using standard reaction condition to furnish pentasaccharide 1
as its 4-methoxyphenyl glycoside. For this purpose a series of suit-
ably functionalized monosaccharide derivatives 2 [13], 3 [14], 4
[15], 5 [16] and 6 [17] were prepared from the commercially avail-
able reducing sugars (Fig. 2).
Stereoselective glycosylation of compound 2 with the thiogly-
coside derivative 3 under iodonium ion mediated thioglycoside
activation condition using N-iodosuccinimide (NIS)–trimethylsilyl
trifluoromethanesulfonate (TMSOTf) [18] furnished disaccharide
derivative 7 in 84% yield, which was deacetylated using sodium
methoxide to give disaccharide diol derivative (8) in quantitative
yield. Appearance of signals at d 5.64 (d, J = 8.5 Hz, H-1A), 5.47 (s,
PhCH), 5.19 (s, PhCH), 4.60 (d, J = 8.0 Hz, 1H, H-1B) in the 1H NMR
and at d 101.1 (C-1B), 100.9 (PhCH), 100.6 (PhCH), 97.9 (C-1A) in
the 13C NMR spectra confirmed the stereoselective formation of
disaccharide derivative 7. In another experiment, compound 4
was treated with N-iodosaccharin in moist acetonitrile [19] to give
hemiacetal derivative, which was converted to trichloroacetimi-
date [20] derivative 9 in 89% yield. Compound 9 was allowed to
glycosylate with thioglycoside acceptor 5 in the presence of
TMSOTf [21] to give disaccharide thioglycoside donor 10 in 77%
yield. In this case thioglycoside 5 has been used as orthogonal gly-
cosyl donor as it acts as a glycosyl acceptor under trichloroacetim-
idate activation condition (Scheme 1).
2.8. 4-Methoxyphenyl (sodium 5-acetamido-3,5-dideoxy-
-galacto-2-nonulopyranosylonate)-(2 ? 6)-(b- -galactopyranosyl)-
(1 ? 6)-(b- -glucopyranosyl)-(1 ? 3)-(b- -galactopyranosyl)-(1 ? 3)-
2-acetamido-2-deoxy-b- -galactopyranoside (1)
D-glycero-a-
D
D
D
D
D
To a solution of the pentasaccharide derivative 13 (700 mg,
0.4 mmol) in methanol (20 mL) was added 20% Pd(OH)2–C
(150 mg) and the reaction mixture was allowed to stir at room
temperature for 24 h under a positive pressure of hydrogen. The
reaction mixture was filtered through a CeliteÒ bed and concen-
trated under reduced pressure. The crude mass was dissolved in
0.1 M sodium methoxide (30 mL) and the reaction mixture was
allowed to stir at room temperature for 12 h and then a few drops
of distilled water was added to the reaction mixture and allowed
to stir for 6 h. The reaction mixture was neutralized with Dowex
50 W X8 (H+) resin, filtered and evaporated to dryness and again
passed through a short pad of Dowex 50 W X8 (Na+) resin. The
crude product was purified by passing through a column of
Sephadex-LH-20 using CH3OH–H2O (4:1) as eluant to give penta-
saccharide 1 as its sodium salt (330 mg, 73%); Rf (0.3, CH3CN–
After preparing a disaccharide glycosyl acceptor 8 and a disac-
charide thioglycoside donor 10, attempts were made towards the
synthesis of target pentasaccharide 1 using stereoselective gly-
cosylations. Iodonium ion mediated regio and stereoselective gly-
cosylation of compound 8 with compound 10 using NIS-TMSOTf
[18] as glycosylation activator furnished (1 ? 3)-b-linked tetrasac-
charide derivative, which was purified after acetylation of the gly-
cosylation product to give the tetrasaccharide derivative 11 in 79%
yield. Presence of signals at d 5.72 (d, J = 8.4 Hz, H-1A), 5.55 (s,
PhCH), 5.33 (s, PhCH), 4.63 (d, J = 7.9 Hz, H-1B), 4.60 (d, J = 8.0 Hz,
H-1D), 4.46 (d, J = 8.0 Hz, H-1C) in the 1H NMR and at d 101.9 (C-
1C), 101.8 (PhCH), 101.5 (C-1D), 100.7 (PhCH), 98.5 (C-1A), 96.1
(C-1B) in the 13C NMR spectra supported the stereoselective forma-
CH3OH–H2O: 2:1:0.5); White powder; ½a D25
ꢁ
ꢀ9.7 (c 1.1, H2O); IR
(neat): 3441, 3021, 1712, 1464, 1230, 767, 669 cmꢀ1
;
1H NMR
(500 MHz, D2O):
d 6.99 (d, J = 8.8 Hz, 2H, Ar-H), 6.86 (d,
J = 8.8 Hz, 2H, Ar-H), 5.09 (d, J = 8.4 Hz, 1H, H-1A), 4.59 (d,
J = 7.8 Hz, 1H, H-1C), 4.48–4.45 (m, 1H, H-5E), 4.29 (d, J = 8.1 Hz,
1H, H-1B), 4.26 (d, J = 7.7 Hz, 1H, H-1D), 4.25–4.22 (m, 2H, H-7E,
H-8E), 4.16–4.12 (m, 1H, H-3A), 4.09–3.87 (m, 5H, H-4A, H-4B,
H-4D, H-4E, H-6E), 3.85–3.59 (m, 12H, H-5D, H-4C, H-2A, H-6abA
,
Ph
Ph
H-6abB, H-9abE, H-6abC, H-6aD), 3.69 (s, 3H, OCH3), 3.57–3.37 (m,
6H, H-5B, H-6bD, H-2D, H-3D, H-2B, H-3B), 3.35–3.22 (m, 4H, H-
2C, H-3C, H-5A, H-5C), 2.67 (dd, J = 12.2, 3.3 Hz, 1H, H-3eE), 1.97
(s, 3H, NHCOCH3), 1.96 (s, 3H, NHCOCH3), 1.69 (t, J = 12.2 Hz,
1H, H-3aE); 13C NMR (125 MHz, D2O): d 175.2 (2C, COONa,
NHCOCH3), 174.6 (NHCOCH3), 118.4–115.5 (Ar-C), 104.1 (C-1C),
104.0 (C-1D), 103.9 (C-1B), 103.8 (C-1A), 101.5 (C-2E), 75.8 (C-
3C), 75.6 (C-3B), 75.4 (C-3A), 74.8 (C-5A), 73.7 (2C, C-3D, C-4E),
73.1 (C-5B), 73.0 (C-5D), 72.8 (C-6E), 71.2 (C-2C), 71.1 (C-5C),
70.8 (C-2B), 70.6 (2C, C-4B, C-4D), 69.3 (2C, C-2D, C-4C), 68.6 (C-
7E), 67.2 (C-4A), 63.6 (C-8E), 63.5 (C-9E), 61.4 (2C, C-6A, C-6C),
61.2 (2C, C-6B, C-6D), 56.2 (OCH3), 52.5 (C-2A), 51.6 (C-5E), 39.4
(C-3E), 22.5 (2C, NHCOCH3); ESI-MS: m/z 1127.5 [M+1]+; Anal.
Calcd for C44H67N2NaO30 (1126.37): C, 46.89; H, 5.99. Found: C,
46.68; H, 6.25.
O
O
O
O
O
O
AcO
SEt
HO
OMp
AcO
NPhth
3
2
OH
AcO
OBn
O
O
AcO
SEt
OAc
AcO
SPh
OAc
AcO
4
5
AcO
OAc
COOCH3
AcO
AcHN
O
Mp=4-methoxyphenyl
Phth=phthaloyl
SPh
AcO
6
Fig. 2. Suitably functionalized monosaccharide intermediates used for the con-
struction of the target pentasaccharide (1).

60
P.K. Mandal, A.K. Misra / Bioorganic Chemistry 38 (2010) 56–61
(PhCH) corresponding to the benzylidene acetal in the 13C NMR
Ph
Ph
O
spectra confirmed the removal of 6-O-benzyl ether leaving benzyl-
idene acetal intact in the compound 12. Finally iodonium ion pro-
moted stereoselective glycosylation of the tetrasaccharide
derivative 12 with sialic acid derived thioglycoside derivative 6
using CH3CN–CH2Cl2 as solvent exploiting nitrile effect [25] of
the solvent afforded pentasaccharide derivative 13 in 53% yield,
in which the N-acetyl neuraminic acid moiety was linked through
O
O
O
a
2 + 3
O
O
A
B
RO
OMp
O
OR
NPhth
7: R = OAc
8: R = H
b
AcO OBn
O
D
AcO
O
AcO
AcO
OBn
O
a
a-(2 ? 6)-linkage. Exclusive formation compound 13 was con-
AcO
5
d
c
4
O
firmed from the signature proton and carbon signals in its NMR
spectra. Prolonged hydrogenolysis followed by saponification of
the pentasaccharide derivative 13 furnished target pentasaccha-
ride 1 as its 4-methoxyphenyl glycoside in 73% yield. Formation
of compound 1 was confirmed from its 1D and 2D NMR and mass
spectral analysis. Signals at d 5.09 (d, J = 8.4 Hz, H-1A), 4.59
(d, J = 7.8 Hz, H-1C), 4.29 (d, J = 8.1 Hz, H-1B), 4.26 (d, J = 7.7 Hz,
H-1D), 4.25–4.22 (m, 2 H, H-7E, H-8E), 2.67 (dd, J = 12.2, 3.3 Hz,
H-3eE), 1.69 (t, J = 12.2 Hz, H-3aE) in the 1H NMR and at d 104.1
(C-1C), 104.0 (C-1D), 103.9 (C-1B), 103.8 (C-1A), 101.5 (C-2E) in
the 13C NMR spectra supported the formation of compound 1
(Scheme 2).
In summary, a concise chemical synthesis of the sialic acid
containing pentasaccharide repeating unit of the O-antigen corre-
sponding to the Shiga toxin producing E. coli O171 as it 4-methoxy-
phenyl glycoside has been carried out successfully involving [2+2]
glycosylation. All glycosylation steps are highly stereoselective and
reproducible for scale-up preparation. Thioglycoside derivative
was used as orthogonally protected glycosyl donor. Regioselective
glycosylation using disaccharide diol acceptor reduced the number
of protecting group manipulation steps. Final stereoselective incor-
poration of N-acetylneuraminic acid (sialic acid) in the target
pentasaccharide derivative was achieved using nitrile effect of
the solvent used in the glycosylation. 4-Methoxybenzyl group
has been chosen as the temporary anomeric protecting group at
the reducing end.
AcO
C
NH
CCl3
SPh
OAc
AcO
AcO
O
10
9
Scheme 1. Reagents: (a) N-iodosuccinimide, TfOH, CH2Cl2, MS-4 Å, ꢀ25 °C, 1 h,
84%; (b) CH3ONa, CH3OH, room temperature, 30 min, quantitative; (c) (i) N-
iodosaccharin, CH3CN–H2O, room temperature, 15 min; (ii) CCl3CN, DBU, CH2Cl2,
5 °C, 6 h, 89% and (d) TMSOTf, CH2Cl2, MS-4 Å, ꢀ20 °C, 1 h, 77%.
tion of tetrasaccharide derivative 11. N-Phthaloyl group of the tet-
rasaccharide derivative 11 was converted to the N-acetyl group on
treatment with ethylenediamine [22] followed by N-acetylation
and the N-acetylated product was subjected to a controlled hydro-
genation condition [12i,23] using H2 over Pearlmans catalyst [24]
to furnish 6-O-debenzylated tetrasaccharide acceptor 12 in 64%
yield having benzylidene acetals unaffected. Besides the ring pro-
tons, presence of two singlets at d 5.56 (s, PhCH), 5.31 (s, 1 H, PhCH)
in the 1H NMR and two signature signals at d 101.9 (PhCH), 100.7
8+10
a,b
AcOOBn
Ph
Ph
O
D
AcO
O
O
O
O
O
AcO
O
O
O
AcO
C
A
B
O
OAc
OMp
NPhth
O
AcO
OAc
Acknowledgments
11
c
P.K.M. thanks CSIR, New Delhi for providing a Senior Research
Fellowship. A.K.M. thanks Department of Science and Technology
(DST), New Delhi for providing Ramanna Fellowship (SR/S1/RFPC-
06/2006).
AcOOH
Ph
Ph
O
D
AcO
O
O
O
O
O
AcO
O
O
O
AcO
A
C
B
O
OMp
NHAc
O
AcO
References
OAc
12
OAc
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AcO
d
6
OAc
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COOCH3
O
AcO
AcHN
E
O
AcO
AcO
Ph
O
Ph
O
D
AcO
O
O
O
O
O
AcO
O
O
O
AcO
B
A
C
O
OMp
NHAc
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OAc
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e
1
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three steps; (d) NIS, TfOH, MS-3 Å, CH3CN–CH2Cl2 (5:1), ꢀ10 °C, 20 h, 53% and (e) (i)
H2, 20% Pd(OH)2–C, CH3OH, room temperature, 24 h; (ii) 0.1 M CH3ONa, CH3OH,
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