Concise synthesis of the tetrasaccharide repeating unit of the O-polysaccharide isolated from Edwardsiella tarda PCM 1156 strain

Article abstract of DOI:10.1016/j.carres.2014.08.004

A convergent strategy has been developed for the synthesis of the tetrasaccharide repeating unit of the O-antigen from Edwardsiella tarda PCM 1156. Sequential glycosylations of a series of rationally protected monosaccharide intermediates were achieved either by the activation of thioglycosides using N-iodosuccinimide (NIS) in conjunction with H₂SO₄-silica or by activation of trichloroacetimidate by H₂SO₄-silica only. All glycosylation reactions resulted in the formation of the desired linkage with absolute stereoselectivity and yielded the required derivatives in good to excellent yields. Both azido and phthalimido groups have been used as the precursor of the desired acetamido group depending on the requirement of 1,2-cis- or 1,2-trans-glycosidic linkage.

Full text of DOI:10.1016/j.carres.2014.08.004

Carbohydrate Research 399 (2014) 15–20
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Concise synthesis of the tetrasaccharide repeating unit of the
O-polysaccharide isolated from Edwardsiella tarda PCM 1156 strain
⇑
Rituparna Das, Mukul Mahanti, Balaram Mukhopadhyay
Indian Institute of Science Education and Research Kolkata, Mohanpur 741246, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2014
Received in revised form 24 July 2014
Accepted 4 August 2014
Available online 21 August 2014
A convergent strategy has been developed for the synthesis of the tetrasaccharide repeating unit of the O-
antigen from Edwardsiella tarda PCM 1156. Sequential glycosylations of a series of rationally protected
monosaccharide intermediates were achieved either by the activation of thioglycosides using N-iodosuc-
cinimide (NIS) in conjunction with H2SO4–silica or by activation of trichloroacetimidate by H2SO4–silica
only. All glycosylation reactions resulted in the formation of the desired linkage with absolute stereose-
lectivity and yielded the required derivatives in good to excellent yields. Both azido and phthalimido
groups have been used as the precursor of the desired acetamido group depending on the requirement
of 1,2-cis- or 1,2-trans-glycosidic linkage.
Keywords:
Bacterial O-antigen
Acetamido sugars
H2SO4–silica
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
as a broad range of other animals.7,8 This species is also responsible
for gastroenteritis, colitis and dysentery like diseases in humans.9
Lipopolysaccharides are the essential and characteristic compo-
nents of the outer membranes of Gram negative bacteria. The O-
polysaccharides are the constituents of these lipopolysaccharides
which are in turn responsible for determining and regulating var-
ious biological conditions of the organisms. The O-antigenic poly-
saccharide chain is made up of oligosaccharide repeating units
and remains exposed on the outer surface of bacterial cell walls.
By virtue of their orientation, the O-polysaccharides are responsi-
ble for binding with the receptors of the host cells and play a piv-
otal role in infections.1 Due to the presence of different sugar
residues, the O-antigens demonstrate a varied character and act
as the elicitor of innate immune responses. There have been exten-
sive studies with these bacterial O-antigens as potential
anti-microbial agents and vaccine-targets.2–4 However, difficult
isolation and cumbersome purification process limit the scope of
accumulating large quantities of these oligosaccharides from natu-
ral sources. Therefore, the chemical synthesis of these O-antigen
repeating units becomes pertinent to explore their vivid biological
roles and potential as vaccine targets.
Several strains of E. tarda including MT108,10 PCM 1153,11 PCM
1150,12 PCM 1145, PCM 1151 and PCM 115813 have been eluci-
dated and studied. Recently, Knirel et al. reported a new structure
of the O-polysaccharide of E. tarda PCM 1156.14 The structural
analysis of this oligosaccharide revealed the presence of a glucosa-
mine, a fucose, a mannose and a galactosamine unit. The biological
implications of the O-antigens of the strain trigger an interest to
build a strategy for the synthesis of the tetrasaccharide repeating
unit. Moreover, the presence of two acetamido sugars in the tetra-
saccharide structure makes it synthetically challenging. Herein, we
report the total chemical synthesis of the tetrasaccharide repeating
unit in the form of its p-methoxyphenyl glycoside (Fig. 1).
2. Results and discussion
Adjudication of the retrosynthetic analysis suggests that
sequential glycosylations of rationally protected monosaccharide
HO
O
Edwardsiella tarda (E. tarda) is a Gram-negative bacterium
belonging to the Endobacteriaceae family.5,6 This microorganism
is an opportunistic pathogen, which is responsible for haemorrhag-
ic septicaemia, also known as Edwardsiellosis among freshwater
fish. It inhabits and infects freshwater and marine fishes as well
HO
OPMP
O
NHAc
O
OH
HO OH
HO
O
O
HO
HO
O
AcNH
1
O
HO OH
⇑
Corresponding author. Tel.: +91 9748 261742; fax: +91 33 25873020.
E-mail address: sugarnet73@hotmail.com (B. Mukhopadhyay).
Figure 1. Structure of the target tetrasaccharide related to the repeating unit of the
O-antigen from E. tarda PCM 1156 in the form of its PMP glycoside.
http://dx.doi.org/10.1016/j.carres.2014.08.004
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

16
R. Das et al. / Carbohydrate Research 399 (2014) 15–20
HO
HO
using TfOH and TMSOTf ended with lower yields, 71% and 74%,
O
OPMP
NHAc
O
respectively, suggesting the better performance of H2SO4–silica.
The phthalimido group was preferred as the acetamido precursor
in order to have 1,2-trans-glycoside at the reducing end. Selective
removal of the chloroacetyl group of the disaccharide 6 using thio-
urea in the presence of 2,4,6-collidine22 yielded the disaccharide
acceptor 7 in 80% yield (Scheme 1).
O
OH
OH
O
HO
HO
HO
O
HO
O
1
AcNH
O
PMP = p-Methoxyphenyl
HOOH
In a separate experiment, the known p-tolyl 2,3,4-tri-O-ben-
zoyl-a-D
-mannopyranoside 823 was treated with chloroacetic
PO
PO
O
OPMP
NHAc
O
PO OP
O
anhydride in the presence of pyridine to get our required mannose
donor 9 in 92% yield. The presence of the benzoyl group in the C-2
position ensures the stereospecific formation of 1,2-trans-glyco-
side. Next, glycosylation of the disaccharide acceptor 7 with the
donor 9 using NIS in the presence of H2SO4–silica at 0 °C yielded
the desired trisaccharide 10 in 82% yield. Selective removal of the
chloroacetyl group using thiourea produced the required trisaccha-
ride acceptor 11 in 80% yield. Final glycosylation between the tri-
saccharide acceptor 11 and the known trichloroacetamide donor
1224 using H2SO4–silica at ꢀ55 °C furnished the protected tetrasac-
charide 13 in 73% yield. Once the protected tetrasaccharide 13 was
obtained, the benzylidene group was selectively cleaved using 80%
acetic acid (AcOH) at 80 °C.25 Then, the azido group was converted
to the desired acetamido functionality by using thioacetic acid26 at
room temperature. Next, the phthalimido group was cleaved using
ethylene diamine27 followed by the global acetylation using acetic
anhydride (Ac2O) in pyridine. Removal of the benzyl groups
through catalytic hydrogenation using Pd–C in the presence of H2
followed by de-O-acetylation using NaOMe in methanol28 yielded
the target tetrasaccharide 1 in 62% yield (Scheme 2).
O
O
CCl3
NH
OP
PO
N3
PO
+
O
PO
O
HO
POOP
Phth = Phthallimido
Commercially available
D-Galactosamine hydrochloride
PO
PO
OP
O
PO
PO
PO
OPMP
O
O
+
NPhth
O
STol
OP
OH
PO
O
Commercially available
D-Mannose
PO
O
PO
HO
STol
OP
OPMP
NPhth
+
OP
PO
Commercially available
L-Fucose
Commercially available
D-Glucosamine hydrochloride
Figure 2. Retrosynthetic analysis for the target tetrasaccharide 1.
synthons are the best bet to achieve the linear tetrasaccharide 1
(Fig. 2). The p-methoxyphenyl glycoside was preferred at the
reducing end of the target 1. It can be cleaved selectively from
the per-O-acetylated derivative of the target tetrasaccharide and
allow the introduction of relevant aglycon using trichloroacetimi-
date chemistry.
3. Conclusion
In conclusion, the total chemical synthesis of the tetrasaccha-
ride repeating unit of the O-antigen from E. tarda has been
achieved by a linear strategy using rationally protected monosac-
charide synthons. The azido precursor was successfully used for
1,2-cis glycosylation whereas the phthalimido precursor lead to
the formation of the required 1,2-trans linkage. Both azido and
phthalimido groups were successfully converted to the desired
acetamido functionality to produce the target tetrasaccharide. Gly-
cosylation reactions were accomplished by our in-house method-
ologies of NIS/H2SO4–silica mediated activation of thioglycosides
or H2SO4–silica mediated activation of glycosyl trichloroacetimi-
dates resulting in good to excellent yield of the required glycosides.
The final target having the PMP glycoside at the reducing end
leaves the scope for its selective removal from the per-O-acety-
lated tetrasaccharide and further conjugation with suitable agly-
con per the biological need.
Thus, the known p-tolyl 2-O-benzyl-1-thio-b-L-fucopyranoside
215 was treated with trimethyl orthobenzoate in the presence of
catalytic amount of 10-camphorsulfonic acid (CSA) to yield the cor-
responding orthobenzoate that was subsequently rearranged by
the treatment of aqueous HCl (1 M)16 to give the required fucose
derivative 3 in 87% yield. The remaining free hydroxyl group in
the 3-position was protected with the chloroacetyl group using
chloroacetic anhydride in the presence of pyridine17 to get the
required fucose donor 4 in 91% yield. Glycosylation between known
acceptor 518 and donor 4 was achieved by the activation of
thioglycoside using N-iodosuccinimide (NIS) in the presence of
H2SO4–silica19–21 at ꢀ40 °C resulting in the required disaccharide
6 in 85% yield. The use of H2SO4–silica for the activation of thiogly-
coside in conjunction with NIS was found to be beneficial over the
traditional use of trifluoromethanesulfonic acid (TfOH) or trimeth-
ylsilyl trifluoromethanesulfonate (TMSOTf) for this purpose. The
solid acid catalyst is easy to handle and can be weighed exactly
per the requirement. It is moisture tolerant and, moreover, the silica
acts as a desiccant to facilitate the anhydrous condition required for
successful glycosylation. Indeed, the same glycosylation reactions
4. Experimental
4.1. General
All solvents and reagents were dried prior to use according to
standardized methods.29 The commercially purchased reagents
Ph
O
O
O
Ph
O
i. Trimethyl
O
OPMP
NPhth
HO
O
orthobenzoate,
OPMP
O
STol
OBn
O
CH3CN, rt
5
STol
OBn
O
NPhth
O
ii. HCl-workup
87%
OR
NIS, H2SO4-silica
OBn
BzO
OH
-40 oC
85%
OH
OR
BzO
Chloroacetic
3 R = H
2
anhydride, py, 0 o
C
4
R = cAc
6 R = cAc
Thiourea, collidine
MeOH-CH2Cl2, reflux
7
R = H
91%
80%
Scheme 1. Synthesis of the disaccharide acceptor 7.

R. Das et al. / Carbohydrate Research 399 (2014) 15–20
17
Ph
O
O
O
OPMP
O
NPhth
Chloroacetic
OBz
O
OBz
O
cAcO
O
HO
BzO
BzO
anhydride, py, 0 o
92%
C
7
OBn
BzO
BzO
BzO
NIS, H2SO4-silica
O
BzO
RO
STol
CH2Cl2, 0 o
82%
C
STol
O
8
9
Thiourea, collidine
MeOH-CH2Cl2
reflux
10 R = cAc
11
BzOOBz
R = H
80%
OAc
O
AcO
O
Ph
O
O
i. 80% AcOH-H2O, 80 o
ii. Thioacetic acid, rt
iii. Ethylene diamine, n-butanol, 110 o
C
O
O
OPMP
O
CCl3
AcO
C
NPhth
N3
O
NH
AcO OAc
O
OBn
12
iv. Ac2O, py, 50 o
C
BzO
v. H2, Pd-C, 50 o
C
AcO
H2SO4-silica
-55 o
73%
O
BzO
vi. NaOMe, MeOH, rt
C
N3
13
O
62%
BzOOBz
HO
HO
O
OPMP
NHAc
O
O
OH
HO OH
HO
O
O
HO
HO
O
AcNH
1
O
HOOH
Scheme 2. Synthesis of the target tetrasaccharide 1.
were used without any further purification unless mentioned
otherwise. Dichloromethane was dried and distilled over P2O5 to
make it anhydrous and moisture-free. All reactions were moni-
tored by thin layer chromatography (TLC) on Silica-Gel 60-F254
with detection by fluorescence followed by charring after immer-
sion in 10% ethanolic solution of H2SO4. Flash chromatography
was performed with silica gel 230–400 mesh. Optical rotations
were measured on a sodium-line at ambient temperature. 1H and
13C NMR were recorded on a Bruker 500 MHz spectrometer. In
the case of the tetrasaccharide, 1H NMR values are denoted as H
for the reducing end glucosamine unit, H0 for the fucose unit, H00
for the mannose unit and H000 for the remaining galactosamine unit.
(2), 129.9(2), 129.6(2), 129.5, 129.3, 128.4(2), 128.3(2), 128.1(2),
127.9 (ArC), 86.8 (C-1), 75.1, 74.0, 73.6, 73.3, 21.1 (S-C6H4CH3)
and 16.7 (C-CH3). HRMS calcd for C27H28O5SNa (M+Na)+:
464.1657, found: 464.1661.
4.4. p-Tolyl 4-O-benzoyl-2-O-benzyl-3-O-chloroacetyl-1-thio-b-
L-fucopyranoside (4)
To a mixture of compound 3 (3.9 g, 8.5 mmol) in dry CH2Cl2
(20 mL), pyridine (5 mL) was added followed by the addition of
chloroacetic anhydride (1.6 g, 9.3 mmol) at 0 °C. The reaction mix-
ture was allowed to stir at the same temperature for 1 h until the
TLC (n-hexane–EtOAc; 2:1) showed complete conversion of the
starting material to a faster moving spot. The solvents were evap-
orated and co-evaporated with toluene. The crude mixture was
dissolved in CH2Cl2 (20 mL) and washed successively with 1 M
HCl (2 ꢁ 25 mL), H2O (25 mL), aqueous NaHCO3 (25 mL) and brine
(25 mL). The organic layer was collected, dried (Na2SO4), filtered
and evaporated in vacuo. The crude mixture thus obtained was
purified by flash chromatography by using n-hexane–EtOAc; 2:1
4.2. Preparation of H2SO4–silica
To a slurry of silica gel 230–400 mesh (10 g) in dry Et2O (20 mL)
was added concentrated H2SO4 (1 mL) and the mixture was hand
shaken for couple of minutes. Then the solvents were evaporated
in vacuo and the residue was dried at 100 °C for 3 h. The dried
material was kept in an airtight bottle for further use.30
as the eluent to yield the pure product 4 (4.1 g, 91%) as a white
25
4.3. p-Tolyl 4-O-benzoyl-2-O-benzyl-1-thio-b-
(3)
L
-fucopyranoside
solid. [a]
ꢀ45 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) d: 8.04–
D
7.19 (m, 14H, ArH), 5.48 (dd, 1H, J3,4 3.0 Hz, J4,51.5 Hz, H-4), 5.19
(dd, 1H, J2,3 10.0 Hz, J3,4 3.0 Hz, H-3), 4.84 (ABq, 1H, JAB11.0 Hz,
CH2Ph), 4.69 (d, 1H, J1,2 10.0 Hz, H-1), 4.57 (ABq, 1H, JAB11.0 Hz,
CH2Ph), 3.91 (m, 1H, H-5), 3.79 (ABq, 2H, JAB 15.0 Hz, COCH2Cl),
3.78 (t, 1H, J1,2J2,3 10.0 Hz, H-2), 2.41 (s, 3H, S-C6H4CH3) and 1.30
(d, 3H, C-CH3). 13C NMR (125 MHz, CDCl3) d: 138.1, 137.9, 133.5,
133.4(2), 129.9(2), 129.7(2), 129.2, 128.7, 128.4(2), 128.3(2),
127.9(2), 127.8 (ArC), 87.0 (C-1), 75.3, 74.5, 73.0, 71.1, 40.4
(COCH2Cl), 21.2 (S-C6H4CH3) and 16.7 (C-CH3). HRMS calculated
for C29H29O6SClNa (M+Na)+: 540.1373, found: 540.1379.
To a suspension of known p-tolyl 2-O-benzyl-1-thio-b-L-fuco-
pyranoside 2 (3.5 g, 9.7 mmol) in dry CH3CN (20 mL), trimethyl
orthobenzoate (2.2 mL, 12.6 mmol) was added followed by the
addition of CSA (25 mg). The reaction mixture was allowed to stir
at room temperature for 2 h until the TLC (n-hexane–EtOAc; 2:1)
showed complete conversion of the starting material. Then the sol-
vents were evaporated in vacuo and the crude mixture was diluted
with CH2Cl2 (25 mL) and washed with 1 M HCl (2 ꢁ 25 mL). The
organic layer was then collected, dried (Na2SO4) and filtered. The
solvents were then evaporated and the crude mixture was purified
4.5. p-Methoxyphenyl 4-O-benzoyl-2-O-benzyl-3-O-
by flash chromatography to give the pure product 3 (3.9 g, 87%) as
chloroacetyl-
deoxy-2-phthalimido-b-D
a
-
L
-fucopyranosyl-(1?3)-4,6-O-benzylidene-2-
-glucopyranoside (6)
25
a colourless syrup. [
a]
ꢀ34 (c 1.0, CHCl3). 1H NMR (500 MHz,
D
CDCl3) d: 8.09–7.21 (m, 14H, ArH), 5.38 (d, 1H, J3,4, J4,5 3.0 Hz, H-
4), 4.96 (ABq, 1H, JAB11.5 Hz, CH2Ph), 4.72 (ABq, 1H, JAB11.5 Hz,
CH2Ph), 4.64 (d, 1H, J1,2 10.0 Hz, H-1), 3.93 (dd, 1H, J2,3 6.0 Hz, J3,4
A mixture of known p-methoxyphenyl 4,6-O-benzylidene-2
deoxy-2-phthalimido-b- -gucopyranoside (1.5 g, 3.0 mmol),
D
5
3.0 Hz, H-3), 3.78 (m, 1H, H-5), 3.67 (t, 1H, J1,2 2,3
J
10.0 Hz, H-2),
donor 4 (1.90 g, 3.5 mmol) and MS 4 Å (2 g) in dry CH2Cl2
(20 mL) was stirred under nitrogen atmosphere for 20 min. Then
NIS (1.1 g, 4.7 mmol) was added followed by the addition of
2.44 (s, 3H, S-C6H4CH3) and 1.31 (d, 3H, C-CH3). 13C NMR
(125 MHz, CDCl3) d: 166.6 (COC6H5), 138.1, 137.7, 133.2, 132.9

18
R. Das et al. / Carbohydrate Research 399 (2014) 15–20
H2SO4–silica (75 mg) and the reaction mixture was stirred at
ꢀ40 °C for 20 min. The TLC (n-hexane–EtOAc 2:1) showed the
complete consumption of the donor 4. The reaction was then neu-
tralized by the addition of Et3N and filtered on a bed of CeliteÒ
(Merck, Germany) and the filtrate was washed successively with
saturated aqueous Na2S2O3 solution (2 ꢁ 25 mL), saturated aque-
ous NaHCO3 solution (2 ꢁ 25 mL) and brine (25 mL). The organic
layer was separated, dried (Na2SO4) filtered and evaporated in
vacuo. The crude mixture obtained was purified by flash chroma-
faster-moving spot. The solvents were evaporated and co-
evaporated with toluene. The crude mixture thus obtained was dis-
solved in CH2Cl2 (20 mL) and washed successively with 1 M HCl
(2 ꢁ 25 mL), H2O (25 mL), aqueous NaHCO3 (25 mL) and brine
(25 mL). The organic layer was collected, dried (Na2SO4), filtered
and evaporated in vacuo. The crude product thus obtained was
purified by flash chromatography by using n-hexane–EtOAc (3:1)
as the eluent to yield the pure 9 (2.0 g, 92%) as a colourless syrup.
25
[a]
+103 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) d: 8.13–7.15
D
tography using n-hexane–EtOAc (2:1) as the eluent to yield the
(m, 19H, ArH), 5.98 (t, 1H, J3,4J4,5 10.0 Hz, H-4), 5.96 (m, 1H, H-2),
5.87 (dd, 1H, J2,3 3.0 Hz, J3,4 10.0 Hz, H-3), 5.71 (d, 1H, J1,2 1.0 Hz,
H-1), 4.9 (m, 1H, H-5), 4.52 (dd, 1H, J5,6a 5.5 Hz, J6a,6b 12.0 Hz, H-
6a), 4.38 (dd, 1H, J5,6b 2.5 Hz, J6a,6b 12.0 Hz, H-6b), 4.04 (ABq, 2H,
JAB 15.0 Hz, COCH2Cl) and 2.34 (s, 3H, S-C6H4CH3). 13C NMR
(125 MHz, CDCl3) d: 166.9 (COCH2Cl), 165.5, 165.4, 165.3 (COC6H5),
138.6, 133.7(2), 133.6(2), 133.3, 132.6(2), 130.1(2), 130.0(2),
129.9(2), 129.8(2), 129.7(2), 129.3, 129.1, 128.6(2), 128.5,
128.4, 128.3 (ArC), 86.1 (C-1), 71.7, 70.1, 69.3, 66.9, 64.2, 40.5
(COCH2Cl) and 21.1 (S-C6H4CH3). HRMS calculated for C36H31O9
ClSNa(M+Na)+: 674.1377, found: 674.1380.
25
disaccharide 6 (2.3 g, 85%) as a white amorphous solid. [
a
]
+53
D
(c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.87–6.74 (m, 23H,
ArH), 5.80 (d, 1H, J1,2 8.5 Hz, H-1), 5.62 (s, 1H, CHPh), 5.31 (m,
2H, H-30, H-40), 4.87 (d, 1H, J1 ,2 3.5 Hz, H-10), 4.82 (dd, 1H, J2,3
10.5 Hz, J3,4 9.0 Hz, H-3), 4.67 (dd, 1H, J1,2 8.5 Hz, J2,3 10.5 Hz, H-
2), 4.45 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 10.5 Hz, H-6a), 4.39 (m, 1H, H-
50), 4.10 (ABq, 1H JAB12.5 Hz, CH2Ph), 3.87 (m, 4H, H-5, H-6b, H-4,
CH2Ph), 3.72 (s, 3H, C6H5OCH3), 3.70 (m, 1H, H-20), 3.56 (dd, 2H,
J15.0 Hz, COCH2Cl) and 0.62 (d, 3H, J 6.5 Hz, C-CH3). 13C NMR
(125 MHz, CDCl3) d: 166.0 (COCH2Cl), 155.5, 150.5, 137.5, 137.0,
133.9, 133.1, 129.6(2), 129.3, 129.2, 128.3(2), 128.2(3), 128.1(3),
127.5, 127.4(2), 126.4(3), 118.5(3), 114.4(3), (ArC), 102.1 (CHPh),
99.3 (C-10), 98.2 (C-1), 81.1, 75.5, 72.7, 72.5, 72.2, 71.8, 68.6, 66.5,
65.2, 55.6, 55.5 (C6H5OCH3), 40.3 (COCH2Cl) and 15.1 (C-CH3).
HRMS calculated for C50H46O14NClNa (M+Na)+: 919.2607, found:
919.2604.
0
0
4.8. p-Methoxyphenyl 2,3,4-tri-O-benzoyl-6-O-chloroacetyl-
a-D-
mannopyranosyl-(1?3)-4-O-benzoyl-2-O-benzyl- -fucopyra
a-L
nosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-phthalim ido-b-D-
glucopyranoside (10)
A mixture of the disaccharide acceptor 7 (1.7 g, 2.0 mmol),
donor 9 (1.75 g, 2.6 mmol) and MS 4 Å (2.0 g) in dry CH2Cl2
(20 mL) was stirred under a nitrogen environment for 20 min. Then
to the reaction mixture, NIS (2.3 g, 3.4 mmol) was added followed
by the addition of H2SO4–silica (75 mg). The reaction mixture was
allowed to stir at 0 °C for 20 min until the TLC (n-hexane–EtOAc
3:1) showed complete conversion of the donor 9. The reaction mix-
ture was immediately filtered on a bed of CeliteÒand the filtrate was
washed successively with saturated aqueous Na2S2O3 (2 ꢁ 25 mL),
saturated NaHCO3 (2 ꢁ 25 mL) and brine (25 mL). The organic layer
was collected, dried (Na2SO4) and filtered, and the solvents were
evaporated in vacuo. The crude product thus obtained was purified
by flash chromatography using n-hexane–EtOAc (2:1) as the eluent
4.6. p-Methoxyphenyl 4-O-benzoyl-2-O-benzyl-a-L-fucopyra
nosyl-(1?3)-4,6-O-benzylidene-2-deoxy-2-phthalim ido-b-D-
glucopyranoside (7)
To a solution of the disaccharide 6 (2.3 g, 2.5 mmol) in MeOH/
CH2Cl2 (3:2; 50 mL), thiourea (1.1 g, 15.0 mmol) was added fol-
lowed by the addition of 2,4,6-collidine (1.7 mL, 12.5 mmol). The
reaction mixture was stirred under reflux for 24 h until the TLC
(n-hexane–EtOAc 1.5:1) showed complete consumption of the
starting material. The reaction mixture was transferred into a sep-
arating funnel, diluted with CH2Cl2 (10 mL) and washed succes-
sively with H2O (3 ꢁ 30 mL). The organic layer was separated,
dried (Na2SO4), filtered and evaporated in vacuo, and the crude
product thus obtained was purified by flash chromatography to
to yield the pure trisaccharide 10 (2.35 g, 82%) as a white amor-
25
phous solid. [
a]
+78 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) d:
D
yield the disaccharide acceptor 7 (1.7 g, 80%) as an amorphous
8.17–6.74 (m, 38H, ArH), 5.92 (d, 1H, J1,2 8.5 Hz, H-1), 5.83 (t, 1H,
white solid. [
a]
D
+65 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) d:
J3 ,4 J4 ,5 10.0 Hz, H-400), 5.64 (s, 1H, CHPh), 5.53 (dd, 1H, J2 ,3
25
00 00
00 00
00 00
3.0 Hz, J3 ,4 10.0 Hz, H-300), 5.48 (m, 2H, H-40, H-200), 4.95 (d, 1H,
00 00
7.85–6.73 (m, 23H, ArH), 5.80 (d, 1H, J1,2 8.5 Hz, H-1), 5.59 (s, 1H,
CHPh), 5.24 (m, 1H, H-40), 4.90 (d, 1H, J1 ,2 3.5 Hz, H-10), 4.81 (dd,
1H, J2,3 10.5 Hz, J3,4 9.0 Hz, H-3), 4.65 (dd, 1H, J1,2 8.5 Hz, J2,3
10.5 Hz, H-2), 4.44 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 10.5 Hz, H-6a), 4.34
(m, 1H, H-50), 4.27 (ABq, 1H JAB12.5 Hz, CH2Ph), 4.13 (m, 1H, H-
30), 3.86 (m, 4H, H-5, H-6b, H-4, CH2Ph), 3.72 (s, 3H, C6H5OCH3),
J1 ,2 3.5 Hz, H-10), 4.64 (d, 1H, J1 ,2 1.5 Hz, H-100), 4.62 (m, 4H, H-
0
0
0
0
00 00
6a, H-3, H-6a00, H-2), 4.46 (m, 3H, H-6b00, H-50, H-500), 4.33 (ABq,
2H, JAB 15.0 Hz, COCH2Cl), 4.24 (ABq, 1H, JAB 12.0 Hz, CH2Ph), 4.19
0
0
0
0
0
(dd, 1H, J2 ,3 10.0 Hz, J3 ,4 3.5 Hz, H-3 ), 3.93 (m, 2H, CH2Ph, H-6b),
3.83 (t, 1H, J3,4J4,5 9.0 Hz, H-4), 3.80 (m, 1H, H-5), 3.74 (dd, 1H,
3.51 (dd, 1H, J1 ,2 3.5 Hz, J2 ,3 10.5 Hz, H-20) and 0.70 (d, 3H, J
6.5 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) d: 166.5 (COC6H5),
155.6, 150.6, 137.5, 137.0, 134.1, 132.9, 131.7, 129.7(2), 129.2,
128.3(3), 128.2(2), 127.8(2), 127.7, 126.2(2), 118.6(2), 114.5(2),
(ArC), 102.0 (CHPh), 98.5 (C-10), 98.2 (C-1), 81.3, 75.0, 74.9, 68.7,
68.3, 66.5, 65.7, 55.9, 55.5 (C6H5OCH3) and 15.6 (C-CH3). HRMS cal-
culated for C48H45O13NNa(M+Na)+: 843.2891, found: 843.2895.
J1 ,2 3.5 Hz, J2 ,3 10.0 Hz, H-30), 3.72 (s, 3H, C6H5OCH3) and 1.03
(d, 3H, J 6.5 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) d: 155.6,
150.5, 137.3, 137.1, 133.8, 133.4, 133.2, 132.9, 131.5, 129.9(2),
129.8(2), 129.7, 129.2(2), 128.9(3), 128.7(2), 128.5(2), 128.2(2),
128.1(2), 127.8, 126.1(2), 118.7(2), 114.5(2)(ArC), 101.4 (CHPh),
99.2 (C-10), 98.5 (C-100), 98.0 (C-1), 82.1, 77.2, 75.5, 73.9, 73.8,
73.0, 69.7, 69.5, 68.7, 68.6, 66.7, 66.4, 66.1, 64.4, 55.5 (C6H5OCH3),
55.3, 40.9 (COCH2Cl) and 16.0 (C-CH3). HRMS calculated for
0
0
0
0
0
0
0
0
4.7. p-Tolyl 2,3,4-tri-O-benzoyl-6-O-chloroacetyl-
a-
D-
C
77H68O22ClNNa(M+Na)+: 1393.3922, found: 1393.3928.
mannopyranoside (9)
4.9. p-Methoxyphenyl 2,3,4-tri-O-benzoyl-a-D-mannopyranos
yl-(1?3)-4-O-benzoyl-2-O-benzyl- -fuco pyranosyl-(1?3)-
4,6-O-benzylidene-2-deoxy-2-phthalimido-b-D-glucopyranoside
(11)
a
-L
To a mixture of p-tolyl 2,3,4-tri-O-benzoyl-a-D-mannopyrano-
side 8 (1.9 g, 3.2 mmol) in dry CH2Cl2 (20 mL), pyridine (5 mL)
was added followed by the addition of chloroacetic anhydride
(600 mg, 3.5 mmol) at 0 °C. The reaction mixture was allowed to
stir at the same temperature for 1 h until the TLC (n-hexane–EtOAc
3:1) showed complete conversion of the starting material to a
To a solution of the trisaccharide 10 (2.35 g, 1.7 mmol) in
MeOH/CH2Cl2 (3:2) (50 mL), thiourea (770 mg, 10.1 mmol) was

R. Das et al. / Carbohydrate Research 399 (2014) 15–20
19
added followed by the addition of 2,4,6-collidine (1.1 mL,
4.11. p-Methoxyphenyl 2-acetamido-2-deoxy-
osyl-(1?6)- -mannopyranosyl-(1?3)-
(1?3)-2-deoxy-2-acetamido-b- -gluco pyranoside (1)
a
-
D
-galactopyran
8.5 mmol). The reaction mixture was stirred under reflux for 48 h
until the TLC (n-hexane–EtOAc 1.5:1) showed complete conversion
of the starting material to a slower moving spot. The mixture was
diluted with CH2Cl2 (10 mL) and washed successively with H2O
(3 ꢁ 30 mL). The organic layer was then separated, dried (Na2SO4)
and filtered, and the solvents were evaporated in vacuo. The crude
product thus obtained was purified by flash chromatography to get
a-D
a-L-fucopyranosyl-
D
Compound 13 (1.6 g, 1.0 mmol) was dissolved in AcOH/H2O
(8:1) (20 mL) and the reaction mixture was stirred at 80 °C for
4 h until the starting material was completely consumed to form
a more polar compound as observed from TLC (n-hexane–EtOAc
1:1). Then the solvents were evaporated and co-evaporated with
toluene. The residue was dried, dissolved in thioacetic acid
(10 mL) and stirred at room temperature in the dark for 72 h, until
the starting material was completely consumed. The solvents were
evaporated and co-evaporated with toluene. The residue was dried
and was further dissolved in n-butanol (15 mL) followed by the
the pure trisaccharide acceptor 11 (1.8 g, 80%) as an amorphous
25
white solid. [
a]
+101 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3)
D
d: 8.16–6.73 (m, 38H, ArH), 5.93 (d, 1H, J1,2 8.5 Hz, H-1), 5.74 (t,
1H, J3 ,4 J4 ,5 10.0 Hz, H-400), 5.69 (dd, 1H, J2 ,3 3.0 Hz, J3 ,4
10.0 Hz, H-300), 5.63 (s, 1H, CHPh), 5.57 (m, 1H, H-40), 5.43 (m,
00 00
00 00
00 00
00 00
1H, H-200), 4.96 (d, 1H, J1 ,2 3.5 Hz, H-10), 4.66 (d, 1H, J1 ,2 1.5 Hz,
0
0
00 00
H-100), 4.61 (m, 2H, H-3, H-2), 4.48 (m, 2H, H-6a, H-50), 4.37 (m,
addition of ethylene diamine (250 lL). The reaction mixture was
1H, H-500), 4.32 (dd, 1H, J2 ,3 3.5 Hz, J3 ,4 10.0 Hz, H-30), 4.21 (ABq,
1H, JAB 12.0 Hz, CH2Ph), 3.93 (m, 4H, CH2Ph, H-6b, H-6a00, H-6b00),
3.78 (m, 3H, H-20, H-4, H-5), 3.71 (s, 3H, C6H5OCH3) and 1.09 (d,
3H, J 6.5 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) d: 155.6, 150.5,
137.3, 137.1, 133.7, 133.4, 133.1, 132.9, 131.3, 130.0(2), 129.9(2),
129.8(2), 129.5(2), 129.3, 129.1(2), 128.9(3), 128.8, 128.5(2),
128.4(2), 128.1(2), 127.8, 126.0(2), 118.7(2), 114.5(2) (ArC), 101.2
(CHPh), 99.1 (C-10), 97.8 (C-1), 97.6 (C-100), 82.3, 77.3, 76.4, 73.7,
73.0, 71.9, 71.2, 70.2, 69.4, 68.6, 67.5, 66.4, 66.3, 62.5, 55.5
(C6H5OCH3), 55.4 and 16.0 (C-CH3). HRMS calculated for
allowed to stir at 110 °C for 24 h until the TLC showed complete
consumption of the starting material. The solvents were evapo-
rated and co-evaporated with toluene and dried. The residue was
dissolved in pyridine (5 mL) followed by the addition of Ac2O
(5 mL) and the mixture was allowed to stir at 50 °C for 10 h. Then
the solvents were evaporated and co-evaporated with toluene. It
was then dissolved in CH2Cl2 (20 mL) and washed successively
with H2O (25 mL). The organic layer was collected, dried over
anhydrous Na2SO4 and filtered. The solvents were evaporated
and the residue was dried. A dilute solution of the residue in MeOH
(30 mL) and AcOH (1 mL) was passed through flow-hydrogenation
assembly fitted with a Pd–C cartridge at 50 °C at normal atmo-
spheric pressure of hydrogen. The starting material was completely
consumed to yield a more polar compound as evident from TLC
(CH2Cl2/MeOH 7:1) after four cycles. The solvents were then evap-
orated in vacuo and the residue was dissolved in MeOH (5 mL) fol-
lowed by the addition of freshly prepared NaOMe in MeOH (5 mL).
The reaction mixture was allowed to stir at 50 °C for 10 h until TLC
(CH2Cl2/MeOH 5:1) showed complete conversion of the starting
material. The reaction was neutralized using DOWEX 50-W H+
resin. The mixture was then filtered through a cotton plug to
remove DOWEX and the filtrate was evaporated in vacuo to yield
0
0
0
0
C
75H67O21NNa (M+Na)+: 1317.4206, found: 1317.4203.
4.10. p-Methoxyphenyl 2,3,4-tri-O-acetyl-2-azido-2-deoxy-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl- -mann opyra-
nosyl-(1?3)-4-O-benzoyl-2-O-benzyl- -fucopyranosyl-(1?3)-
a-D-
a-D
a-L
4,6-O-benzylidene-2-deoxy-2-phthali mido-b-D-glucopyranoside
(13)
A mixture of trisaccharide acceptor 11 (1.8 g, 1.4 mmol), donor
12 (536 mg, 1.6 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (15 mL)
was allowed to stir at ꢀ55 °C under a nitrogen atmosphere for
30 min. H2SO4–silica (50 mg) was added to the reaction mixture
and it was allowed to stir at the same temperature for 12 h. Then
the reaction was quenched by adding Et3N and immediately fil-
tered over a bed of CeliteÒ. The solvents were evaporated in vacuo
and the crude product was purified by flash chromatography using
the pure tetrasaccharide 1 (510 mg, 62%) as a white sticky com-
25
pound. [
a]
+36 (c 0.8, CH3OH). 1H NMR (500 MHz, CD3OD) d:
D
0
0
6.95, 6.82 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.11 (d, 1H, J1 ,2 1.5 Hz,
H-10), 4.96 (d, 1H, J1,2 8.5 Hz, H-1), 4.94 (d, 1H, J1
3.5 Hz, H-
000,2000
n-hexane–EtOAc (2:1) to give the pure tetrasaccharide 13 (1.6 g,
1000), 4.85 (s, 1H, H-100), 3.73 (s, 3H, C6H4OCH3), 2.01, 1.98 (2s, 6H,
25
73%) as an amorphous solid. [
a
]
+82 (c 1.0, CHCl3). 1H NMR
2 ꢁ NHCOCH3) and 1.26 (d, 3H,
J
6.5 Hz, C-CH3). 13C NMR
D
(500 MHz, CDCl3) d: 8.20–6.74 (m, 38H, ArH), 5.95 (d, 1H, J1,2
(125 MHz, CD3OD) d: 174.6, 174.4 (2 ꢁ NHCOCH3), 156.8, 153.1,
119.2(2), 115.6(2) (ArC), 103.8 (C-10), 101.8 (C-1), 101.6
(C-1000), 99.1 (C-100), 83.0, 79.3, 78.1, 73.5, 73.2, 72.5, 72.4, 72.0,
70.4, 70.3, 70.2, 69.1, 68.3, 68.2, 67.5, 62.7, 62.5, 56.9, 56.1 (C6H4-
OCH3), 52.6, 51.8, 23.0, 22.6 (2 ꢁ NHCOCH3) and 16.6 (C-CH3).
HRMS calculated for C35H54O21N2Na (M+Na)+: 861.3117, found:
861.3121.
8.5 Hz, H-1), 5.84 (t, 1H, J3 ,4 J4 ,5 10.0 Hz, H-400), 5.82 (dd, 1H,
J2 11.0 Hz, J3
3.0 Hz, H-3000), 5.66 (s, 1H, CHPh), 5.65
00 00
00 00
000,3000
000,4000
(m, 1H, H-4000), 5.59 (dd, 1H, J2 ,3 3.0 Hz, J3 ,4 10.0 Hz, H-300), 5.56
00 00
00 00
(d, 1H, J3 ,4 3.5 Hz, J4 ,5 <1.0 Hz, H-40) , 5.49 (dd, 1H, J1 ,2 1.5 Hz,
0
0
0
0
00 00
J2 ,3 3.0 Hz, H-200), 5.21 (d, 1H, J1 ,2 3.5 Hz, H-10), 4.90 (d, 1H,
00 00
0
0
3.0 Hz, H-1000), 4.75 (d, 1H, J1 ,2 1.5 Hz, H-100), 4.73 (m, 1H,
00 00
000,2000
J1
H-500), 4.61 (m, 3H, H-2, H-3, H-50), 4.47 (dd, 1H, J5,6a 4.5 Hz, J6a,6b
10.5 Hz, H-6a), 4.32 (dd, 1H, J2 ,3 10.0, Hz, J3 ,4 3.5 Hz, H-30), 4.15
(m, 2H, CH2Ph, H-6a00), 4.06 (m, 3H, H-6a000, H-6b00, H-4), 3.90 (t,
1H, J5,6b Hz, J6a,6b 12.0 Hz, H-6b), 3.77 (m, 5H, H-6b000, CH2Ph, H-
5000, H-2000, H-5), 3.72 (s, 3H, C6H5OCH3), 3.70 (m, 1H, H-20), 2.16,
2.14, 1.84 (3s, 9H, 3 ꢁ COCH3) and 1.04 (d, 3H, J 6.5 Hz, C-CH3).
13C NMR (125 MHz, CDCl3) d: 170.3, 169.9, 169.5 (COCH3), 166.8
(COCH2Cl), 165.6, 164.9, 164.8 (COC6H5), 155.4, 150.6, 137.5,
137.2, 133.6(2), 133.3, 133.0, (3), 132.8, 131.6, 130.0(3), 129.9(2),
129.8(2), 129.5(2), 129.3, 129.2, 129.1, 128.8, 128.4(2), 128.2(2),
128.1(2), 127.9(2), 127.7(2), 126.1(3), 122.9, 118.5(2), 114.4(2)
(ArC), 101.2 (CHPh), 99.4(C-1000), 98.3 (C-100), 98.0 (C-10), 97.6 (C-
1), 81.7, 77.5, 76.1, 74.0, 73.0, 72.8, 70.0, 69.7, 69.5, 68.7, 68.5,
68.1, 67.1, 66.8, 66.7, 66.3, 66.2, 62.2, 57.9, 55.6 (C6H5OCH3), 55.2
and 15.8 (C-CH3). HRMS calculated for C87H82O28N4Na(M+Na)+:
1630.5116, found: 1630.5119.
0
0
0
0
Acknowledgments
R.D. and M.M. are thankful to IISER Kolkata for Senior Research
Fellowship and Student fellowship respectively. This work is sup-
ported by the Research Grant SB/S1/OC-48/2013 from SERB, DST,
New Delhi to B.M.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.
08.004.

20
R. Das et al. / Carbohydrate Research 399 (2014) 15–20
15. Budhadev, D.; Mukhopadhyay, B. Carbohydr. Res. 2014, 394, 26–31.
References
16. Mukhopadhyay, B.; Field, R. A. Carbohydr. Res. 2003, 338, 2149–2152.
17. Bertolini, M. J.; Glaudemans, C. P. J. Carbohydr. Res. 1970, 15, 263–270.
18. Kocharova, N. A.; Kondakova, A. N.; Vinogradov, E.; Ovchinnikova, O. G.;
Lindner, B.; Shashkov, A. S.; Rozalski, A.; Knirel, Y. A. Chem. Eur. J. 2008, 14,
6184–6191.
19. Das, R.; Mukhopadhyay, B. Carbohydr. Res. 2013, 376, 1–6.
20. Verma, P. R.; Mukhopadhyay, B. RSC Adv. 2013, 3, 201–207.
21. Pal, K. B.; Mukhopadhyay, B. Carbohydr. Res. 2013, 379, 26–29.
22. Takeo, K.; Maki, K.; Wada, Y.; Kitamura, S. Carbohydr. Res. 1993, 245, 81–96.
23. Kowalczyk, R.; Harris, P. W. R.; Dunbar, R. P.; Brimble, M. A. Synthesis 2009, 13,
2210–2222.
24. Grunler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984, 1826–1847.
25. Gent, P. A.; Gigg, R. J. Chem. Soc., Perkin Trans. 1 1974, 1446–1455.
26. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996, 292, 71–81.
27. Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Carbohydr. Res. 1993, 243,
139–164.
1. Weintraub, A. Carbohydr. Res. 2003, 338, 2539–2547.
2. Roy, R. Drug Discovery Today: Technol. 2004, 1, 327–336.
3. Doores, K. J.; Gamblin, D. P.; Davis, B. G. Chem.-Eur. J. 2006, 12, 656–665.
4. Kuberan, B.; Linhardt, R. J. Curr. Org. Chem. 2000, 4, 653–677.
5. Ewing, W. H.; McWhorter, A. C.; Escobar, M. R.; Lubin, A. H. Int. Bull. Bacteriol.
Nomencl. Taxon. 1965, 15, 33–38.
6. Grimont, P. A. D.; Grimont, F.; Richard, C.; Sakazaki, R. Curr. Microbiol. 1980, 4,
347–351.
7. Janda, J. M.; Abbott, S. L. Clin. Infect. Dis. 1993, 17, 742–748.
8. Mohanty, B. R.; Sahoo, P. K. J. Biosci. 2007, 32, 1331–1344.
9. Abbott, S. L.; Janda, J. M. Prokaryotes 2006, 6, 72–89.
10. Vinogradov, E.; Nossova, L.; Perry, M. B.; Kay, W. W. Carbohydr. Res. 2005, 340,
85–90.
11. Katzenellenbogen, E.; Kocharova, N. A.; Toukach, P. V.; Górska, S.; Bogulska, M.;
Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2012, 355, 56–62.
12. Katzenellenbogen, E.; Kocharova, N. A.; Shashkov, A. S.; Górska-Fra˛czek, S.;
Bogulska, M.; Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2013, 368, 84–88.
13. Katzenellenbogen, E.; Kocharova, N. A.; Toukach, P. V.; Gorska, S.; Bogulska, M.;
Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2012, 347, 186–188.
14. Katzenellenbogen, E.; Kocharova, N. A.; Shashkov, A. S.; Górska-Fra˛czek, S.;
Gamian, A.; Knirel, Y. A. Carbohydr. Res. 2013, 374, 45–48.
28. Zemplén, G. Ber. Deutch. Chem. Ges. 1926, 59, 1254–1266.
29. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification of Laboratory Chemicals;
Pergamon: London, 1996.
30. Mukhopadhyay, B. Tetrahedron Lett. 2006, 47, 4337–4341.