Chemical synthesis of a tetrasaccharide related to the repeating unit of the O-polysaccharide isolated from Salmonella enterica O59

Article abstract of DOI:10.1016/j.tetasy.2011.11.016

The chemical synthesis of the tetrasachharide repeating unit of the O-polysaccharide of Salmonella enterica O59 in the form of its p-methoxyphenyl glycoside is reported. The total synthesis of the tetrasaccharide has been achieved through concise protecti

Full text of DOI:10.1016/j.tetasy.2011.11.016

Tetrahedron: Asymmetry 22 (2011) 2007–2011
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemical synthesis of a tetrasaccharide related to the repeating unit of the
O-polysaccharide isolated from Salmonella enterica O59
⇑
Priya Verma, Balaram Mukhopadhyay
Indian Institute of Science Education and Research-Kolkata (IISER-K), Mohanpur Campus, PO BCKV Campus Main Office, Mohanpur, Nadia 741252, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemical synthesis of the tetrasachharide repeating unit of the O-polysaccharide of Salmonella enter-
ica O59 in the form of its p-methoxyphenyl glycoside is reported. The total synthesis of the tetrasaccha-
ride has been achieved through concise protecting group manipulations and stereoselective
glycosylations via thioglycoside activation using La(OTf)3 as a Lewis acid promoter in conjunction with
N-iodosuccinimide. La(OTf)3 was found to be a better alternative to the more traditional Lewis acid pro-
moters such as TfOH or TMSOTf.
Received 11 October 2011
Accepted 30 November 2011
Available online 3 January 2012
Dedicated to Professor Ulf J. Nilsson, Lund
University
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
-β-D-Galp-(1
3)-α-D-GlcpNAc-(1
4)-α-L-Rhap-(1
OH
3)-β-D-GlcpNAc-(1
Bacterial O-antigens (O-polysaccharides), constituents of the
lipopolysaccharides on the cell walls of Gram-negative bacteria,
are important for the pathogenicity of the bacteria and responsible
for interactions with the host immune response.1 O-Antigens are
one of the most diverse classes of molecules present in the bacte-
rial cell wall due to genetic variations in the O-antigen gene clus-
ters. Due to the antigenic character, bacterial O-antigens in the
form of glyco-conjugates are potential targets for the development
of anti-bacterial agents or vaccines. There are already reports of
such kind in the literature.2 However, it is impossible to collect
adequate amounts of material for making suitable glyco-conju-
gates and detailed biological studies to evaluate their potential
use. Therefore, a synthetic procedure is required for the large-scale
production of the O-antigen structure from commercially available
sugars.
O
HO
OMP
O
NHAc
Me
OH
HO
O
O
AcHN
O
HO
O
HO
OH
HO
O
OH
OH
1
MP = p-methoxyphenyl
Figure 1. Structure of the repeating unit of the O-antigen from S. enterica and the
synthetic target.
2. Results and discussion
Salmonella enterica, the causative agent for salmonellosis, is a
major pathogen of humans and animals. It is responsible for
millions of cases of food-related diseases and an alarming number
of deaths in the US per annum.3 On the basis of the diverse
O-antigens present in the lipopolysaccharide, strains of S. enterica
are classified into 46 O-serogroups. The structures of the O-anti-
gens of many of them have been elucidated.4 Recently, Perepelov
et. al.5 reported the structure of the tetrasachharide repeating unit
of the O-antigen isolated from the lipopolysaccharide of S. enterica
O59 containing a galactose, a rhamnose and two glucosamine
moieties (Fig. 1). Herein, we report the total synthesis of the tetra-
saccharide repeating unit of the O-antigen from S. enterica in the
form of its p-methoxyphenyl glycoside.
The p-methoxyphenyl group was preferred as the reducing end
glycoside due to its chemical stability during various protecting
group manipulations and glycosylation reactions. Moreover, it
can be removed selectively from the acylated/arylated target struc-
ture and allow trichloroacetimidate chemistry for further conjuga-
tion with suitable aglycons. A sequential glycosylation strategy
using acetals as permanent protecting groups and acyls as tempo-
rary protecting groups was assumed to be suitable for the synthe-
sis of the target tetrasaccharide 1. Therefore, four suitably
protected monosaccharide synthons 2–5 were prepared following
literature procedures (Fig. 2).
Glycosylation between acceptor 26 and donor 37 was achieved
by activation of the thioglycoside with N-iodosuccinimide (NIS)
in the presence of La(OTf)38 giving the disaccharide 6 in 86% yield.
Solid, moisture tolerant La(OTf)3 was proven to be a better alterna-
tive to the more conventional Lewis acids such as TfOH or TMSOTf.
⇑
Corresponding author. Tel.: +91 9748 261742; fax: +91 33 2587 3031.
E-mail address: sugarnet73@hotmail.com (B. Mukhopadhyay).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.11.016

2008
P. Verma, B. Mukhopadhyay / Tetrahedron: Asymmetry 22 (2011) 2007–2011
8 in 78% yield, exclusively as the
a-anomer. De-O-acetylation of
TolS
the trisaccharide 8 gave the trisaccharide acceptor 9 in 86% yield.
Final glycosylation between trisaccharide acceptor 9 and known
monosaccharide donor 512 using NIS-La(OTf)3 mediated thioglyco-
side activation afforded the protected tetrasaccharide 10 in 83%
yield. The N-phthalimido group was converted to an N-acetyl
derivative using ethylene diamine in n-butanol13 followed by acet-
ylation using Ac2O in pyridine to afford tetrasaccharide 11 in 76%
yield. Next, tetrasaccharide 11 was treated with thiolacetic acid14
to convert the azido functionality to desired N-acetyl. Global de-
protection was achieved by hydrolysis of the acetals using 80% ace-
tic acid at 80 °C15 followed by de-O-acetylation with NaOMe in
MeOH to give the target tetrasaccharide 1 in 78% overall yield
(Scheme 1).
O
Ph
Ph
Me
AcO
O
O
O
OMP
HO
O
NPhTh
O
2
3
OAc
O
AcO
AcO
O
O
O
STol
AcO
OAc
N3
4
STol
5
Figure 2. Suitably protected monosaccharide synthons.
3. Conclusion
Similar glycosylation reactions with TfOH9 and TMSOTf afforded
the desired disaccharide in 56% and 65% yield, respectively, and
partial hydrolysis of the benzylidene acetal was observed in both
cases. De-O-acetylation of disaccharide 6 in the presence of NaOMe
in MeOH10 afforded disaccharide acceptor 7 in 87% yield. Further
glycosylation of acceptor 7 with monosaccharide donor 411 was
accomplished by similar NIS-La(OTf)3 mediated activation at
ꢀ50 °C. The reaction proceeded smoothly to afford trisaccharide
The chemical synthesis of the tetrasaccharide repeating unit of
the O-antigen from S. enterica in the form of its p-methoxyphenyl
glycoside has been accomplished by rational protecting group
manipulations and stereoselective glycosylations. The final prod-
uct is suitable for further conjugation with the desired aglycon to
form glyco-conjugates. Further studies in this direction are cur-
rently ongoing and the results will be reported in due course.
O
Ph
O
O
4
OMP
O
NIS, La(OTf)3
NIS, La(OTf)3
NPhTh
2 + 3
-50 oC, 78%
0 oC, 86%
Me
RO
O
O
O
6 R = Ac
7 R = H
NaOMe, MeOH
87%
O
Ph
O
O
OMP
NPhTh
O
5
NIS, La(OTf)3
Me
O
O
0 oC, 83%
N3
O
RO
O
O
O
Ph
O
8 R = Ac
9 R = H
NaOMe, MeOH
86%
O
O
O
Ph
O
OMP
AcO
i. ethylene diamine, 110 oC, 24h
ii. Ac2O, Py
OAc
NPhTh
Me
O
AcO
O
O
N3
O
48h, 76%
O
O
OAc
O
O
Ph
O
10
O
O
Ph
O
O
OMP
NHAc
AcO
OAc
Me
O
i. thiolacetic acid
O
AcO
1
O
N3
ii. NaOMe, MeOH
78%
O
O
O
OAc
O
O
Ph
O
11
Scheme 1. Synthesis of the tetrasaccharide 1.

P. Verma, B. Mukhopadhyay / Tetrahedron: Asymmetry 22 (2011) 2007–2011
2009
0
0
J5,6a 10 Hz, J6a,6b 10.5 Hz, H-6a), 4.03 (dd, 1H, J1 ,2 1.9 Hz, J
4. Experimental
0
0
0
0
0
2 ,3 5.5 Hz, H-2 ), 3.88–3.75 (m, 5H, H-6b, H-5 , H-3 , H-4, H-5),
3.72 (s, 3H, C6H4OCH3), 2.00 (s, 3H, COCH3), 1.3 (s, 3H, C-CH3),
4.1. General methods
1.03 (s, 3H, C-CH3), 0.57 (d, 3H, J5 ,6 6.0 Hz, H-60). 13C NMR (CDCl3,
125 MHz) d: 169.92 (COCH3), 155.69, 150.45, 136.94, 134.53, (2),
129.23, 129.01, 128.58, 128.94, 128.20 (2), 128.13, 126.96 (2),
123.78, 118.72 (2), 114.5 (2), 109.46 (isopropylidene-C), 102.15
(CHPh), 98.05 (C-1), 97.49 (C-10), 80.39, 75.76, 75.46, 74.04,
73.84, 68.69, 66.57, 64.59, 56.61, 55.56 (C6H4OCH3), 27.36, 26.05
(2 ꢂ C–CH3), 20.93 (COCH3), 16.03 (C-60). HRMS cald for
0
0
All glycosylation reactions were carried out in septum-capped,
oven dried round bottom flasks. N2 gas was purged to make the
reaction conditions free of moisture. Dichloromethane was dried
and distilled over P2O5 to make it moisture free. All reactions were
monitored by Thin layer chromatography (TLC) on Silica gel 60-
F254 with detection by fluorescence followed by charring after
immersion in 10% ethanolic solution of H2SO4. All reagents and sol-
vents were dried prior to use according to standard methods.16
Commercial reagents were used without further purification un-
less otherwise stated. Flash chromatography was performed with
Silica Gel 230–400 mesh. Optical rotations were measured on so-
dium D-line at ambient temperature. 1H and 13C NMR spectra were
recorded on Bruker 500 MHz Spectrometer. In case of tetrasaccha-
ride, 1H NMR values are denoted as H for reducing end Glucosa-
mine, H0 for Rhamnose unit and H00 for 2nd Glucosamine unit
and H000 for the Galactose unit.
C
39H41NO13Na (M+Na)+: 754.2476, found: 754.2469.
4.1.3. p-Methoxyphenyl 2,3-O-isopropylidene-
rhamnopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-N-
phthalimido-b- -glucopyranoside 7
a-L-
D
To a solution of disaccharide 6 (1.25 g, 1.7 mmol) in MeOH
(10 mL) was added NaOMe in MeOH (0.5 M, 1.0 mL) and the solu-
tion was stirred at room temperature for 30 min. After completion
of the reaction, it was neutralized with DOWEX 50 W H+ resin and
filtered through a cotton plug. The solvent was evaporated in vacuo
and the crude material was purified by flash chromatography using
n-hexane–EtOAc (1:1) as eluent to afford pure disaccharide accep-
4.1.1. p-Tolyl 3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-1-
tor 7 (1.0 g, 87%) as a white foam. ½a D25
ꢁ
¼ þ132 (c 1.1, CHCl3). 1H
thio-
The known p-tolyl 2-azido-4,6-O-benzylidene-2-deoxy-1-thio-
-glucopyranoside (4.80 g, 12.0 mmol) was dissolved in pyridine
a-D-glucopyranoside 4
NMR (CDCl3, 500 MHz) d: 7.87–7.33 (m, 9H, ArH), 6.8, 6.7 (2d,
4H, C6H4OCH3), 5.82 (d, 1H, J1,2 8.5 Hz, H-1), 5.55 (s, 1H, CHPh),
4.71 (s, 1H, H-10), 4.70 (m, 1H, H-3), 4.55 (dd, 1H, J1,2 9.0 Hz, J2,3
10.0 Hz, H-2), 4.42 (dd, 1H, J5,6a 10.0 Hz, J6a,6b 10.5 Hz, H-6a), 3.92
(t, 1H, H-6b), 3.87–3.72 (m, 5H, H-20, H-30, H-40, H-50, H-4), 3.70
(s, 3H, C6H4OCH3), 3.14 (m, 1H, H-5), 2.52 (bs, 1H, OH), 1.26 (s,
a-D
(5 mL) followed by the addition of Ac2O (5 mL) and the solution
was stirred at room temperature for 2 h until the completion of
the reaction was evident from TLC (n-hexane–EtOAc; 3:1). Solvents
were evaporated using co-evaporation with toluene under reduced
pressure. This residue was dissolved in CH2Cl2 (20 mL) and washed
successively with H2O (20 mL) and brine (20 mL). The organic layer
was collected, dried (Na2SO4) and filtered. The solvents were evap-
orated under reduced pressure and the crude material was purified
by flash chromatography to give compound 4 (5.10 g, 97%) as a col-
3H, C–CH3), 0.71 (d, 3H, J5 6 6.5 Hz, H-60). 13C NMR (CDCl3,
125 MHz) d: 155.56, 150.37, 136.83, 134.39 (2), 131.13, 129.11,
128.11 (2), 126.35 (2), 123.64 (2), 118.63 (2), 114.41 (2), 108.98
(isopropylidene-C), 102.02 (CHPh), 97.95 (C-1), 97.83 (C-10),
80.24, 78.05, 75.66, 74.21, 74.20, 68.53, 66.55, 66.23, 56.57 (C-6),
55.45 (C6H4OCH3), 27.60, 25.69 (2 ꢂ C–CH3), 16.44 (C-60). HRMS
cald for C37H39NO12Na (M+Na)+: 712.2370, found: 712.2362
0
0
ourless gel. ½a 2D5
ꢁ
¼ þ112 (c 1.1, CHCl3). 1H NMR (CDCl3, 500 MHz)
d: 7.49–7.36 (m, 7H, ArH), 7.15 (d, 2H, C6H4CH3), 5.57 (d, 1H, J1,2
5.5 Hz, H-1), 5.53–5.49 (m, 2H, H-3, CHPh), 4.55 (m, 1H, H-5),
4.25 (d, 1H, J5,6a 10.0 Hz, J6a,6b 10.5 Hz, H-6a), 4.06 (dd, 1H, J1,2
5.5 Hz, J2,3 10.5 Hz, H-2), 3.79 (t, 1H, J5,6b 10.0 Hz, J6a,6b 10.5 Hz,
H-6b), 3.69 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 2.35 (s, 3H, C6H4SCH3),
2.16 (s, 3H, COCH3). 13C NMR (CDCl3, 125 MHz) d: 169.33 (COCH3),
138.35, 136.71, 133.05 (2), 129.90 (2), 128.98, 128.68, 128.11 (2),
126.01 (2), 101.49 (CHPh), 87.87 (C-1), 79.40, 70.35, 68.32, 63.66,
62.69, 21.01 (C6H4SCH3), 20.66 (COCH3). HRMS calcd for
4.1.4. p-Methoxyphenyl 3-O-acetyl-2-azido-4,6-O-benzylidene-
2-deoxy-
rhamnopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-N-
phthalimido-b- -glucopyranoside 8
a-D-glucopyranosyl-(1?4)-2,3-O-isopropylidene-a-L-
D
A mixture of disaccharide acceptor 7 (1.0 g, 1.5 mmol), donor 4
(775 mg, 1.75 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (15 mL) was
stirred under nitrogen for 30 min. Then NIS (525 mg, 2.3 mmol)
was added followed by La(OTf)3 (50 mg) and the mixture was stir-
red at ꢀ50 °C for 45 min when TLC (n-hexane–EtOAc, 2:1) showed
complete conversion of the starting materials to form a new com-
pound. The mixture was filtered through a pad of Celite, washed
with CH2Cl2 and the combined filtrate was washed successively
with aq. Na2S2O3 (2 ꢂ 25 mL), saturated NaHCO3 (2 ꢂ 25 mL) and
brine (25 mL). The organic layer was collected, dried (Na2SO4)
and filtered, and the solvents were evaporated in vacuo. The crude
residue was purified by flash chromatography using n-hexane–
EtOAc (3:1) to afford pure trisaccharide 8 (1.15 g, 78%) as a white
C
22H23N3O5SNa (M+Na)+: 464.1256, found: 464.1251
4.1.2. p-Methoxyphenyl 4-O-acetyl-2,3-O-isopropylidene-
rhamnopyranosyl-(1?3)-4,6-O-benzylidene-2-deoxy-N-
phthalimido-b-D-glucopyranoside 6
a-L-
A mixture of acceptor 2 (1.0 g, 2.0 mmol), donor 3 (840 mg,
2.4 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (15 mL) was stirred un-
der nitrogen for 30 min. Then NIS (710 mg, 3.1 mmol) was added
followed by La(OTf)3 (50 mg) and the mixture was stirred at room
temperature for 45 min when TLC (n-hexane-EtOAc; 2:1) showed
complete consumption of the donor 3. The mixture was filtered
through a pad of Celite, washed with CH2Cl2 and the combined fil-
trate was washed successively with aq Na2S2O3 (2 ꢂ 25 mL), satu-
rated NaHCO3 (2 ꢂ 25 mL) and brine (25 mL). The organic layer
was collected, dried (Na2SO4) and filtered, and the solvents were
evaporated in vacuo. The crude residue was purified by flash chro-
matography using n-hexane–EtOAc (3:1) to afford pure disaccha-
foam. ½a 2D5
ꢁ
¼ þ142 (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz) d:
7.87–7.30 (m, 14H, ArH), 6.87–6.75 (2dd, 4H, C6H4OCH3), 5.84 (d,
00 00
1H, J1,2 8.5 Hz, H-1), 5.56 (s, 1H, CHPh), 5.54 (t, 1H, J2 ,3 10.0 Hz,
J3 ,4 10.0 Hz, H-300), 5.46 (s, 1H, CHPh), 4.86 (d,1H, J1 ,2 4.0 Hz,
H-100), 4.68 (s, 1H, H-10), 4.64 (dd, 1H, J1,2 8.5 Hz, J2,3 10.5 Hz, H-
00 00
00 00
00
00
00
00
2), 4.42 (m, 1H, H-6b), 4.19 (dd, 1H, J5 ,6a 10.0 Hz, J6a ,6b
10.0 Hz, H-6a00), 4.11 (m, 1H, H-40), 3.99 (m, 1H, H-50), 3.86–3.76
ride 6 (1.25 g, 86%) as a white foam. ½a D25
¼ þ127 (c 1.0, CHCl3).
ꢁ
(m, 5H, H-6a, H-50, H-20, H-30, H-4), 3.72 (s, 3H, C6H4OCH3), 3.66
(t, 1H, J5 ,6b 9.5 Hz, J6a 6b 10.0 Hz, H-6b00), 3.58 (t, 1H, J3 ,4
1H NMR (CDCl3, 500 MHz) d: 7.34–7.89 (m, 9H, ArH), 6.84, 6.82
(2dd, 4H, C6H4OCH3), 5.82 (d, 1H, H-1), 5.57 (s, 1H, CHPh), 4.75
(s, 1H, H-10), 4.74 (m, 1H, H-3), 4.63 (dd, 1H, H-40), 4.55 (dd, 1H,
00
00
00
00
00 00
10.0 Hz, J4 ,5 9.5 Hz, H-400), 3.16–3.09 (m, 2H, H-200, H-5), 2.14 (s,
00 00
0
0
3H, COCH3), 1.25,1.04 (s, 3H, 2 ꢂ C-CH3), 0.70 (d, 3H, J5 ,6 6.0 Hz,

2010
P. Verma, B. Mukhopadhyay / Tetrahedron: Asymmetry 22 (2011) 2007–2011
H-60). 13C NMR (CDCl3, 125 MHz) d: 169.81, (COCH3), 155.68,
150.49, 136.96, 138.7, 134.53 (2), 131.25, 129.28, 129.08, 128.24
(3), 128.20 (2), 126.67 (2), 126.17 (2), 123.74 (2), 118.79 (2),
114.50 (2), 108.79 (isopropylidene-C), 102.49, 101.56 (2 ꢂ CHPh),
99.04 (C-100), 98.04 (C-1), 97.69 (C-10), 81.09, 80.35, 79.29, 76.16,
75.75, 74.59, 68.84, 68.70, 66.73, 65.31, 62.50, 61.86, 56.58, 55.57
(C6H4OCH3), 53.41, 27.80, 25.94 (2 ꢂ C–CH3), 20.86 (COCH3),
16.60 (C-60). HRMS cald for C52H54N4O17Na (M+Na)+: 1029.3382,
found: 1029.3389.
1H, H-6a), 4.20–3.97 (m, 6H, H-300, H-40, H-500, H-6a000, H-6b000, H-
6a00), 3.86 (m, 6H, H-20, H-30, H-6b, H-400, H-4, H-50), 3.70 (s, 3H,
C6H4OCH3), 3.65–3.63 (m, 1H, H-6b00), 3.23 (m, 1H, H-200), 3.10
(m, 1H, H-50), 2.15–1.91 (3s, 12H, 4 ꢂ COCH3), 1.25, 1.01 (2s, 6H,
2 ꢂ C-CH3), 0.69 (d, 3H, J5 ,6 6.0 Hz, H-60). 13C NMR (CDCl3,
125 MHz) d: 170.24, 170.20, 170.07, 169.49 (4 ꢂ COCH3), 155.72,
150.46, 134.51 (2), 131.24, 130.86, 129.31, 129.26, 129.18,
129.01, 128.78, 128.33 (2), 128.30 (2), 126.64 (2), 126.62 (2),
126.52 (2), 123.74, 118.78 (2), 114.51 (20, 108.85 (isopropyli-
dene-C), 102.44, 101.42 (2 ꢂ CHPh), 101.58 (C-100), 98.79 (C-1000),
98.04 (C-1), 97.79 (C-10), 80.54, 80.38, 76.22, 75.88, 74.85, 71.11,
70.64, 69.35, 68.82, 68.71, 66.72, 66.69, 65.38, 65.34, 62.98,
62.58, 56.63, 55.57 (C6H4OCH3), 27.82, 25.98 (2 ꢂ C–CH3), 20.81,
20.75, 20.63, 20.56 (4 ꢂ COCH3), 16.68 (C-60). HRMS cald for
0
0
4.1.5. p-Methoxyphenyl 2-azido-4,6-O-benzylidene-2-deoxy-
-glucopyranosyl-(1?4)-2,3-O-isopropylidene-
rhamnopyranosyl-(1?3)-2-deoxy-N-phthalimido-4,6-O-
benzylidene-b- -glucopyranoside 9
a-
D
a-L-
D
To a solution of the trisaccharide 8 (1.15 g, 1.14 mmol) in MeOH
(10 mL) was added NaOMe in MeOH (0.5 M, 1.0 mL) and the reac-
tion was stirred at room temperature for 30 min. After completion
of the reaction, it was neutralized with DOWEX 50 W H+ resin and
filtered through the cotton plug. The solvents were evaporated in
vacuo and the crude material was purified by flash chromatogra-
phy using n-hexane–EtOAc (1:1) as eluent to afford pure com-
C
64H70N4O25Na (M+Na)+: 1317.4227, found: 1317.4222.
4.1.7. p-Methoxyphenyl 2,3,4,6-tetra-O-acetyl-b-
osyl-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
osyl-(1?4)-2,3-O-isopropylidene- -rhamnopyranosyl-(1?3)-
D
-galactopyran
a-D
-glucopyran
a-L
2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-glucopyranoside
11
pound 9 (950 mg, 86%) as a white foam. ½a D25
ꢁ
¼ þ148 (c 1.0,
To a solution of compound 10 (1.05 g, 0.8 mmol) in n-butanol
(10 mL) was added ethylene diamine (0.2 mL) and the reaction
mixture was allowed to stir at 110 °C for 24 h. The solvents were
removed under reduced pressure and a solution of the crude prod-
uct in pyridine (5 mL) was treated with acetic anhydride (5 mL) at
room temperature for 3 h. The reaction mixture was evaporated
and co-evaporated with toluene and passed through a short pad
of SiO2 using n-hexane–EtOAc (1:1) as eluent to give the pure com-
CHCl3). 1H NMR (CDCl3, 500 MHz) d: 7.88–7.16 (m, 14H, ArH),
6.85–6.75 (2dd, 4H, C6H4OCH3), 5.84 (d, 1H, J1,2 8.5 Hz, H-1), 5.56
(s, 1H, CHPh), 5.51 (s, 1H, CHPh), 4.81 (d, 1H, J1 ,2 4.0 Hz, H-100),
00 00
4.67 (s, 1H, H-10), 4.66 (m, 1H, H-3), 4.55 (dd, 1H, J1,2 8.5 Hz, J2,3
10.0 Hz, H-2), 4.43 (dd, 1H, J5,6a 10.5 Hz, J6a,6b 10.5 Hz, H-6a),
4.19–4.14 (m, 2H, H-300, H-6a00), 4.04–3.99 (m, 2H, H-40, H-500),
3.86 (m, 1H, H-6b), 3.80–3.76 (m, 4H, H-30, H-20, H-4, H-50), 3.7
(s, 3H, C6H4OCH3), 3.66 (t, 1H, J5 ,6b 10.0 Hz, J6a 6b 10.5 Hz, H-
pound 11 (740 mg, 76%) as colourless syrup. ½a D25
¼ þ117 (c 0.9,
ꢁ
00
00
00
00
6b00), 3.49 (t, 1H, J3 ,4 9.5 Hz, J4 ,5 9.5 Hz, H-400), 3.20 (dd, 1H, J1 ,2
CHCl3). 1H NMR (CDCl3, 500 MHz) d: 7.49–7.32 (m, 10H, ArH),
6.88–6.74 (2d, 4H, C6H4OCH3), 6.52 (bd, 1H, NHAc), 5.52, 5.42
(2s, 2H, 2 ꢂ CHPh), 5.36–5.23 (m, 2H, H-4000, H-2000), 5.15 (d, 1H,
J1,2 8.5 Hz, H-1), 5.06 (s, 1H, H-10), 4.99 (m, 1H, H-3000), 4.85 (d,
00 00
00 00
00 00
00 00
4.0 Hz, J2 ,3 10.0 Hz, H-200), 3.13 (dd, 1H, H-5), 2.68 (bs, 1H, OH),
00 00
1.27, 1.08 (s, 3H, 2 ꢂ C-CH3), 0.69 (d, 3H, J5 ,6 6.0 Hz, H-60). 13C
NMR (CDCl3, 125 MHz) d: 155.69, 150.48, 137.85, 136.94, 136.87,
134.49 (2), 131.25, 129.34, 129.02, 128.37 (2), 128.22 (2), 126.62
(2), 126.26 (2), 125.28, 123.74, 118.77 (2), 114.51 (2), 108.80 (iso-
propylidene-C), 102.49, 102.02 (2 ꢂ CHPh), 98.82, (C-1), 98.03 (C-
100), 97.75 (C-10), 81.71, 80.89, 80.34, 76.29, 75.89, 74.73, 68.75,
68.71, 68.64, 66.71, 65.42, 63.20, 62.13, 56.66, 55.57 (C6H4OCH3),
27.82, 26.00 (2 ꢂ C–CH3), 16.62 (C-60). HRMS cald for C50H52N4O16-
Na (M+Na)+: 987.3276, found: 987.3271.
0
0
1H, J1 ,2 3.0 Hz, H-100), 4.73, (d, 1H, J1
8.0 Hz, H-1000), 4.32 (m,
000,2000
1H, H-6a), 4.20–4.03 (m, 8H, H-6b000, H-6a000, H-6a00, H-40, H-500, H-
300, H-2, H-3), 3.84 (m, 3H, H-6b, H-400, H-50), 3.70 (s, 3H, C6H4OCH3),
3.67 (m, 4H, H-4, H-6b00, H-20, H-30), 3.23 (m, 2H, H-200, H-5), 2.16–
1.90 (s, 15H, 5 ꢂ COCH3), 1.40, 1.24 (s, 6H, 2 ꢂ C–CH3), 0.75 (d, 3H,
J5 ,6 5.5 Hz, H-60). 13C NMR (CDCl3, 125 MHz) d: 170.66, 170.25,
170.18, 170.09, 169.39, 155.45, 150.90, 137.02, 129.15, 129.09,
128.23, 128.20, 128.14, 128.02, 126.46, 126.26, 126.03, 125.90,
1125.80, 118.59 (2), 114.59, 114.46, 108.94 (isopropylidene-C),
102.03 (CHPh), 101.44 (C-1000), 101.35 (CHPh), 100.09 (C-1), 98.63
(C-100), 97.79 (C-10), 80.94, 80.25, 79.55, 76.07, 76.03, 71.00,
70.85, 70.49, 69.25 69.12, 68.73, 68.63, 68.53, 66.63, 66.34, 65.06,
63.94, 62.87, 62.69, 62.52, 62.38, 60.60, 60.32, 57.30, 57.15, 55.46
(C6H4OCH3), 28.02, 26.26, (2 ꢂ C–CH3), 23.19 (NHCOCH3), 20.53,
20.47, 20.44, 20.39 (4 ꢂ COCH3), 14.07 (C-60). HRMS cald for
C58H70N4O24Na (M+Na)+: 1229.4278, found: 1229.4272.
0
0
4.1.6. p-Methoxyphenyl 2,3,4,6-tetra-O-acetyl-b-
osyl-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
osyl-(1?4)-2,3-O-isopropylidene- -rhamnopyranosyl-(1?3)-
D-galactopyran
a-D-glucopyran
a
-L
4,6-O-benzylidene-2-deoxy-N-phthalimido-b-D-
glucopyranoside 10
A mixture of trisaccharide acceptor 9 (950 mg, 0.984 mmol), do-
nor 5 (540 mg, 1.2 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (15 mL)
was stirred under nitrogen for 30 min. Then NIS (360 mg,
1.6 mmol) was added followed by La(OTf)3 (50 mg) and the mix-
ture was stirred at room temperature for 45 min when TLC (n-hex-
ane–EtOAc, 2:1) showed complete conversion of the starting
materials to form a new compound. The mixture was filtered
through a pad of Celite, washed with CH2Cl2 and the combined fil-
trate was washed successively with aq Na2S2O3 (2 ꢂ 25 mL), satu-
rated NaHCO3 (2 ꢂ 25 mL) and brine (25 mL). The organic layer
was collected, dried (Na2SO4) and filtered, and the solvents were
evaporated in vacuo. The crude residue was purified by flash chro-
matography using n-hexane–EtOAc (3:1) to afford pure tetrasac-
4.1.8. p-Methoxyphenyl b-
ido-2-deoxy- -glucopyranosyl-(1?4)-
(1?3)-2-acetamido-2-deoxy-b- -glucopyranoside 1
D-galactopyranosyl-(1?3)-2-acetam
a-
D
a-L
-rhamnopyranosyl-
D
Compound 11 (740 mg, 0.6 mmol) was dissolved in thiolacetic
acid (5 mL) and this was stirred at room temperature for 48 h. After
the completion of the reaction, the thiolacetic acid was co-evapo-
rated with toluene and the residue was washed with NaHCO3
(2 ꢂ 50 mL). The organic layer was separated, dried (Na2SO4) and
filtered. The solvent was evaporated and the residue was dried.
This was treated with 80% AcOH (10 mL) and stirred at 80 °C until
the TLC showed complete conversion of the starting material. The
solvent was co-evaporated with toluene and the residue was dried.
The residue thus obtained was dissolved in MeOH (10 mL), fol-
lowed by the addition of NaOMe in MeOH (0.5 M, 1 mL) and the
solution was stirred at room temperature for 4 h. After completion
charide 10 (1.05 g, 83%) as a white foam. ½a D25
¼ þ103 (c 0.9,
ꢁ
CHCl3). 1H NMR (CDCl3, 500 MHz) d: 7.87–7.35 (m, 14H, ArH),
6.85–6.75 (2d, 4H, C6H4OCH3), 5.84 (d, 1H, J1,2 8.5 Hz, H-1), 5.58,
5.56 (2s, 2H, 2 ꢂ CHPh), 5.37–5.24 (m, 2H, H-4000,H-2000), 4.82 (d,
00 00
1H, J1 ,2 3.5 Hz, H-100), 4.75 (d, 1H, J1
8.0 Hz, H-1000), 4.67 (m,
000,2000
2H, H-10, H-3), 4.54 (t, 1H, J1,2 8.5 Hz, J2,3 9.5 Hz, H-2), 4.42 (m,

P. Verma, B. Mukhopadhyay / Tetrahedron: Asymmetry 22 (2011) 2007–2011
2011
of the reaction, it was neutralized with DOWEX 50 W H+ resin and
filtered through the cotton plug. Solvents were evaporated in va-
cuo to afford pure target tetrasaccharide 1 (400 mg, 78%) as white
2. (a) Roy, R. Drug Discovery Today: Technol. 2004, 1, 327–336; (b) Tamborrini, M.;
Werz, D. B.; Frey, J.; Pluschke, G.; Seeberger, P. H. Angew. Chem., Int. Ed. 2006,
45, 6581–6582; (c) Verez-Bencomo, V.; Fernández-Santana, V.; Hardy, E.;
Toledo, M. E.; Rodríguez, M. C.; Heynngnezz, L.; Rodriguez, A.; Baly, A.; Herrera,
L.; Izquierdo, M.; Villar, A.; Valdés, Y.; Cosme, K.; Deler, M. L.; Montane, M.;
Garcia, E.; Ramos, A.; Aguilar, A.; Medina, E.; Toraño, G.; Sosa, I.; Hernandez, I.;
Martínez, R.; Muzachio, A.; Carmenates, A.; Costa, L.; Cardoso, F.; Campa, C.;
Diaz, M.; Roy, R. Science 2004, 305, 522–525; (d) Snippe, H.; van Dam, J. E. G.;
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Immun. 1983, 42, 842–844.
solid. ½a 2D5
ꢁ
¼ þ81 (c 0.8, H2O). 1H NMR (D2O, 500 MHz) d: 6.94, 6.85
00 00
(2d, 4H, C6H4OCH3), 4.97 (d, 1H, J1,2 8.5 Hz, H-1), 4.88 (d, 1H, J1 ,2
3.5 Hz, H-100), 4.76 (s, 1H, H-10), 4.34 (d, 1H, J1
8.0 Hz, H-1000),
000,2000
4.01–3.31 (m, 25H, H-20, H-30, H-40, H-50, H-200, H-300, H-400, H-500,
H-6a00, H-6b00, H-2, H-3, H-4, H-5, H-6a, H-6b, H-2000, H-3000, H-4000,
H-5000, H-6a000, H-6b000, C6H4OCH3), 1.93, 1.91 (2s, 6H, 2 ꢂ COCH3),
3. Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, J. S.; Shapiro, C.; Griffin,
P. M.; Tauxe, R. V. Emerg. Infect. Dis. 1999, 5, 607–625.
1.13 (d, 3H, J5 6 6.0 Hz, C-CH3). 13C NMR (D2O, 125 MHz) d:
174.58, 174.33 (2 ꢂ COCH3), 154.93, 150.84, 118.46 (2), 115.08
(2), 103.37 (C-1000), 101.13 (C-10), 99.92 (C-1), 98.01 (C-100), 81.42,
80.56, 76.17, 76.11, 75.27, 72.53, 71.52, 70.93, 70.71, 68.88,
68.57, 68.43, 68.28, 67.99, 61.02, 61.05, 60.09, 55.8 (C6H4OCH3),
55.34, 52.76, 21.88, 21.84 (2 ꢂ NHCOCH3) 16.80 (C-60). HRMS calcd
for C35H54N2O21Na (M+Na)+: 861.3117, found: 861.3123.
0
0
4. (a) Knirel, Y. A.; Kochetkov, N. K. Biochemistry (Moscow) 1994, 59, 1325–1383;
Jansson, P.-E. In Endotoxin in Health and Disease; Brade, H., Opal, S. M., Vogel, S.
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Knirel, Y. A.; Wang, L. Carbohydr. Res. 2011, 346, 381–383.
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2172–2176.
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1331.
Acknowledgements
10. Zemplén, G.; Gerecs, A.; Hadácsy, I. Ber. Dtsch. Chem. Ges. 1936, 69, 1827–1830.
11. p-Tolyl
2-azido-4,6-O-benzylidene-2-deoxy-1-thio–
D
-glucopyranoside
is
PV is thankful to University Grants Commission (UGC), New
Delhi for a Senior Research Fellowship. This work is supported by
Department of Science and Technology, New Delhi through re-
search grant to BM (SR/S1/OC-67/2009).
reported in: L. Huang, X. Huang, Chem. Eur. J. 2007, 13, 529-540. Acetylation
of the 3-OH is described in the experimental section in details.
12. Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.; Field, R. A. J. Org. Chem. 2004,
69, 7758–7760.
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