Chemical synthesis of the tetrasaccharide repeating unit of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565

Article abstract of DOI:10.1016/j.carres.2013.04.033

Chemical synthesis of the tetrasaccharide repeating unit of the O-antigenic polysaccharide from Plesiomonas shigelloides strain AM36565 is reported. Glycosylations between suitably protected monosaccharide synthons were achieved by the activation of thiog

Full text of DOI:10.1016/j.carres.2013.04.033

Carbohydrate Research 376 (2013) 1–6
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemical synthesis of the tetrasaccharide repeating unit of the
O-antigenic polysaccharide from Plesiomonas shigelloides strain
AM36565
⇑
Rituparna Das, Balaram Mukhopadhyay
Indian Institute of Science Education and Research-Kolkata (IISER-K), Mohanpur Campus, P.O. 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:
Chemical synthesis of the tetrasaccharide repeating unit of the O-antigenic polysaccharide from Plesiomo-
nas shigelloides strain AM36565 is reported. Glycosylations between suitably protected monosaccharide
synthons were achieved by the activation of thioglycosides in the presence of H2SO4–silica in conjunction
with N-iodosuccinimide. The glycosylations accomplished were highly stereoselective and afforded the
desired products in good to excellent yields.
Received 13 March 2013
Received in revised form 22 April 2013
Accepted 30 April 2013
Available online 7 May 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Oligosaccharide
Total synthesis
Shigellosis
H2SO4–silica
1. Introduction
been determined and elucidated.3–6 Recently, Sawen et al.7 eluci-
dated the structure of the tetrasaccharide repeating unit O-anti-
The O-polysaccharide constituents of many pathogenic and non-
pathogenic bacterial species play diverse roles in determining and
regulating the biology of the organisms.1 The bacterial O-antigens,
constituents of the lipopolysaccharides of the bacterial cell wall,
consist of many repeats of the oligosaccharide units. Taking advan-
tage of the presence of a variety of sugar residues, these O-antigens
are considered to be one of the most diverse classes of molecules
present on the bacterial cell wall. They play a key-role as an elicitor
of innate immune responses.2 They also contribute to the pathoge-
nicity by virtue of their protecting ability of the infecting bacteria by
killing serum complement and phagocytosis.2 Due to this antigenic
character of bacterial O-antigens, they are widely studied as poten-
tial vaccine-targets. However, isolation of these oligosaccharides
from natural sources in adequate quantity and purity is almost
impossible. Thus, synthetic methods are employed to explore their
potential for future carbohydrate-based vaccines.
Plesiomonas shigelloides is a Gram-negative, facultative anaerobe
that causes enteric disease in human, especially following the con-
sumption of raw seafood. They are generally found in fresh or estu-
arine waters as saline environment restricts their growth. Hence, it
has been found to be responsible for various outbreaks of diarrheal
disease associated with contaminated water. To date, only a few of
the O-antigenic polysaccharide structures of P. shigelloides have
genic polysaccharide from P. shigelloides strain AM36565.
Structural analysis of the oligosaccharide showed that it consists
of a glucosamine, a galactosamine and two rhamnose moieties.
Herein, we report the total synthesis of the tetrasaccharide repeat-
ing unit in the form of its p-methoxyphenyl glycoside (Fig. 1)
through suitable protecting group manipulations on the commer-
cially available monosaccharides followed by stereoselective
chemical glycosylations.
2. Results and discussion
With the aim of designing the synthetic strategy for the effec-
tive synthesis of the target tetrasaccharide, p-methoxyphenyl
group was selected at the reducing end as it leaves the possibility
of further glycoconjugate formation by selective removal of the
glycoside from the per-O-acetylated form of the target tetrasaccha-
ride 1. Retrosynthetic analysis of 1 suggests a sequential glycosyl-
ation strategy with suitably protected monosaccharide synthons
(Fig. 2).
Thus, the known p-methoxyphenyl 2-azido-2-deoxy-a-D-gluco-
pyranoside 28 was selectively protected at the C-4 and C-6 posi-
tions by benzaldehyde dimethyl acetal9 in the presence of 10-
camphorsulfonic acid (CSA) to form the required acceptor 3 in
83% yield. The selection of azido group as the acetamido precursor
is essential to have 1,2-cis-glycoside at the reducing end. Then the
acceptor 3 was coupled to known thioglycoside donor 410 having
⇑
Corresponding author. Tel.: +91 9748 261742; fax: +91 33 25873020.
E-mail address: sugarnet73@hotmail.com (B. Mukhopadhyay).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.04.033

2
R. Das, B. Mukhopadhyay / Carbohydrate Research 376 (2013) 1–6
ing of the benzylidene acetal using NaCNBH3 and HCl–ether18 or
triethylsilane and BF3ꢀEt2O19 to form 4-OH acceptor failed to afford
the desired product in reasonable yield. Further, 2-OH position of
the known p-tolyl 3,4-di-O-benzyl-1-thio-a-L-rhamnopyranoside
(8)20 was protected with chloroacetyl group using chloroacetic
anhydride in the presence of pyridine21 to yield p-tolyl 3,4-di-O-
benzyl-2-O-chloroacetyl-1-thio-a-L-rhamnopyranoside (9) in 85%
isolated yield. Presence of the chloroacetate group at C-2 is justi-
fied as it will ensure the 1,2-trans glycosylation through neighbor-
ing group participation and it is orthogonal to the acetates present
in the disaccharide acceptor.
Glycosylation between the disaccharide acceptor
7 and
rhamnosyl donor 9 was accomplished by activating the thioglyco-
side with NIS in the presence of H2SO4–silica furnishing the trisac-
charide 10. Purification of the trisaccharide 10 was unsuccessful as
traces of the unreacted acceptor contamination remained. Selec-
tive removal of the chloroacetate group using thiourea22 afforded
the trisaccharide acceptor 11 in 78% yield. Final glycosylation of
the trisaccharide acceptor 11 with the known p-tolyl 2,3,4-tri-O-
Figure 1. Structure of the tetrasaccharide repeating unit and the target tetrasac-
charide as its p-methoxyphenyl glycoside.
phthalimido as the acetamido precursor to facilitate required 1,2-
trans glycosylation through neighboring group participation. Acti-
vation of the thioglycoside using NIS in the presence of H2SO4–sil-
acetyl-1-thio-a-L
-rhamnopyranoside (12)23 using the similar thio-
glycoside activation furnished the protected tetrasaccharide 13 in
80% yield.
ica11–15 afforded the desired disaccharide
5 in 85% yield
Once the tetrasaccharide framework is established, the next
challenge was to convert the acetamido precursors into the desired
acetamido forms. First, the azido group was converted into acet-
amido using thioacetic acid by a slow reaction for 72 h at ambient
temperature in the dark24 to give the corresponding acetamido
derivative 14 in 72% yield. Next, the phthalimido group was re-
moved by ethylenediamine25 and the free amine thus formed,
was acetylated by using Ac2O and pyridine. It is worth mentioning
that the benzoyl group along with other acetate groups was re-
moved by the action of ethylenediamine and subsequently acety-
lated during acetylation of the free amine. Next, the removal of
the benzyl groups was accomplished by catalytic hydrogenation
using Pd–C in the presence of H2. Finally, the acetates were re-
moved by Zemplén de-O-acetylation26 using NaOMe in methanol
to afford the target tetrasaccharide 1 in 74% yield.
stereospecifically as evident from NMR spectra. Hydrolysis of the
benzylidene acetal using 80% AcOH16 at 80 °C furnished the diol
6 in 94% yield. Owing to the greater reactivity of the primary OH
group, it was regioselectively benzoylated using BzCN in the pres-
ence of Et3N17 to afford the required disaccharide acceptor 7 in 88%
yield. It is worth noting that the obvious choice of reductive open-
OH
HO
HO
O
O
HO
HO
O
OH
O
OH
HO
O
O
O
HO
AcNH
3. Conclusion
NHAc
OMP
MP =
p
-Methoxyphenyl
In summary, we have achieved the chemical synthesis of the
tetrasaccharide repeating unit of the O-antigenic polysaccharide
from Plesiomonas shigelloides strain AM36565 in the form of its p-
methoxyphenyl derivative through rational protecting group
manipulations and stereoselective glycosylations using thioglyco-
sides activated by H2SO4–silica in conjunction with NIS. Further,
the p-methoxyphenyl group can be cleaved selectively from the
per-O-acetylated tetrasaccharide and conjugated with specific
aglycon using trichloroacetimidate chemistry (Scheme 1).
OH
PO
PO
STol
O
OP
O
PO
O
+
OP
PO
O
O
O
PO
OP
PO
N3
OMP
NPhth
Phth = Phthallimido
Commercially available
-Rha
L
4. Experimental section
4.1. General methods
STol
OP
OP
O
PO
PO
O
HO
+
O
PO
O
N3
OMP
PO
OP
NPhth
All solvents and reagents were dried prior to use according to
literature methods.27 The commercially purchased reagents were
used without any further purification unless mentioned otherwise.
Dichloromethane wad dried and distilled over P2O5 to make it
anhydrous and moisture-free. All reactions were monitored by
Thin Layer Chromatography (TLC) on Silica Gel 60-F254 with detec-
tion by fluorescence followed by charring after immersion in 10%
ethanolic solution of H2SO4. Flash chromatography was performed
with Silica Gel 230–400 mesh. Optical rotations were measured on
sodium D-line at ambient temperature. 1H and 13C NMRs were re-
corded on Bruker 500 MHz spectrometer. In the case of the tetra-
saccharide, 1H NMR values are denoted as H for the reducing end
OP
Commercially available
-Rha
STol
PO
PO
OP
O
O
L
PO
HO
+
N3
OMP
NPhth
Commercially available
-GlcNH2.HCl and
-GalNH2.HCl
D
D
Figure 2. Retrosynthetic analysis for the synthesis of the target tetrasaccharide 1.

R. Das, B. Mukhopadhyay / Carbohydrate Research 376 (2013) 1–6
3
OH
O
Benzaldehyde
dimethylacetal
O
Ph
O
O
HO
HO
HO
CSA
83%
N3
N3
OMP
2
OMP
3
OAc
AcO
AcO
O
O
Ph
STol
O
O
OAc
AcO
AcO
80% AcOH
80 o
94%
O
NPhth
O
4
N3
C
OMP
NPhth
NIS, H2SO4-silica
85%
5
OH
O
OAc
HO
OBz
AcO
AcO
OAc
O
AcO
AcO
BzCN
O
HO
O
O
Et3N
88%
O
N3
N3
NPhth
OMP
NPhth
OMP
6
STol
7
O
BnO
OCA
BnO
BnO
BnO
AcO
OR
O
OBz
O
8 R = H
Chloroacetic
anhydride, Py
OAc
O
Thiourea
78%
O
9 R = CA
O
85%
AcO
N3
NIS, H2SO4-silica
NPhth
OMP
10
STol
OH
BnO
O
AcO
BnO
AcO
O
OBz
O
AcO
12
OAc
OAc
O
O
O
AcO
N3
NIS, H2SO4-silica
80%
NPhth
OMP
11
OAc
OAc
AcO
AcO
AcO
AcO
O
O
1) Ethylenediamine
2) Ac2O, Py
Thioacetic acid
72%
O
O
BnO
BnO
BnO
AcO
BnO
AcO
O
O
OBz
O
OAc
O
OAc
OAc
O
O
O
O
O
O
AcO
N3
AcO
AcNH
NPhth
13
OMP
OMP
NPhth
14
OAc
OH
AcO
HO
AcO
HO
O
O
1. H2, Pd-C
O
O
BnO
BnO
AcO
HO
HO
HO
2. NaOMe, MeOH
74%
O
OAc
O
O
OH
O
OAc
OH
O
O
O
O
O
O
AcO
AcNH
HO
AcNH
OMP
AcNH
OMP
NHAc
15
1
Scheme 1. Synthesis of the tetrasaccharide (1).
glucosamine unit, H0 for the galactosamine unit, H00 for the rham-
nose unit attached to the glucosamine unit, and H000 for the remain-
ing rhamnose unit.
and the residue was dried at 100 °C for 3 h. The dried material was
kept in an airtight bottle for further use.
4.3. p-Methoxyphenyl 2-azido-4,6-O-benzylidiene-2-deoxy-a-D-
4.2. Preparation of H2SO4–silica
glucopyranoside (3)
To slurry of silica gel 230–400 mesh (10 g) in dry Et2O (20 mL)
was added concd. H2SO4 (1 mL) and the mixture was hand shaken
for couple of minutes. Then the solvents were evaporated in vacuo
To a mixture of known p-methoxyphenyl 2-azido-2-deoxy-a-D-
glucopyranoside (2)8 (3.0 g, 9.6 mmol) in dry acetonitrile (30 mL)
was added benzaldehyde dimethyl acetal (2.2 mL, 14.5 mmol) fol-

4
R. Das, B. Mukhopadhyay / Carbohydrate Research 376 (2013) 1–6
0
0
0
0
0
lowed by a catalytic amount of CSA at room temperature. The pH
was checked till optimum acidity (pH ꢁ3 to 4) was attained. The
reaction mixture was allowed to stir for 2 h until the TLC (n-hex-
ane/EtOAc; 1:1) showed complete conversion of the starting mate-
rial. Then the solution was neutralized with Et3N, the solvent was
evaporated in vacuo and the crude mixture thus obtained was
purified by flash chromatography using n-hexane/EtOAc (2:1) as
the eluent to afford pure compound 3 (3.2 g, 83%) as white amor-
11.5 Hz, J3 ,4 3.5 Hz, H-30), 5.51 (d, 1H, J3 ,4 , 3.5 Hz, H-4 ), 5.47 (d,
1H, J1 ,2 8.5 Hz, H-10), 5.31 (d, 1H, J1,2 3.5 Hz, H-1), 4.62 (dd, 1H,
0
0
J1 ,2 8.5 Hz, J2 ,3 11.5 Hz, H-20), 4.25 (m, 3H, H-6a0, H-50, H-6a), 3.9
(dd, 1H, J2,3 11.0 Hz, J3,4 9.0 Hz, H-3), 3.8 (m, 3H, H-5, H-6b0, H-
6a), 3.73 (s, 3H, C6H4OCH3), 3.64 (t, 1H, J3,4 J4,5 9.0 Hz, H-4), 3.12
(dd, 1H, J1,2 3.5 Hz, J2,3 11.0 Hz, H-2), 2.21, 2.07, 1.87 (3s, 9H,
3 ꢃ COCH3), 1.25 (s, 2H, OH). 13C NMR (125 MHz, CDCl3) d: 170.4,
170.1, 169.6 (3 ꢃ COCH3), 168.3, 167.6 (2 ꢃ phthalimido CO),
134.3, 134.2, 131.6, 131.3, 123.6, 123.5, 118.1(3), 114.7(3) (ArC),
99.8 (C-10), 97.4 (C-1), 55.6 (C6H4OCH3), 20.6, 20.5, 20.4, (3 x
COCH3). HRMS calcd for C33H36N4O15 (M+Na)+: 728.2177, found:
728.2181.
0
0
0
0
phous mass. ½a 2D5
ꢂ
+103° (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3)
d: 7.51–6.85 (m, 9H, ArH), 5.56 (s, 1H, CHPh), 5.45 (d, 1H, J1,2
3.5 Hz, H-1), 4.41(t, 1H, J2,3, J3,4 9.5 Hz, H-3), 4.26 (dd, 1H, J5,6b
5.0 Hz, J6a,
10.0 Hz, H-6b), 4.06 (m, 1H, H-5), 3.79 (s, 3H,
6b
C6H4OCH3), 3.76 (m, 1H, H-6a), 3.6 (t, J3,4, J4,5 9.5 Hz, H-4), 3.4
(dd, 1H, J1,2 3.5 Hz, J2,3 9.5 Hz, H-2), 2.95 (br s, 1H, OH). 13C NMR
(125 MHz, CDCl3) d: 129.4, 128.4(2), 126.3(3), 118.2(2), 114.7,
114.6 (ArC), 102.2 (CHPh), 98.3 (C-1), 81.7 (C-4), 68.7 (C-3), 68.6
(C-6), 63.0 (C-5), 62.9 (C-2), 55.6 (C6H4OCH3). HRMS calcd for
4.6. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-
D-galactopyranosyl-(13)-6-O-benzoyl-2-azido-2-
deoxy- -glucopyranoside (7)
a
-D
C
20H21N3O6 (M+Na)+: 399.3972, found: 399.3976.
To a solution of compound 6 (1.6 g, 2.2 mmol) in CH3CN
(15 mL), BzCN (260 L, 2.2 mmol) was added followed by Et3N
(50 L) and the mixture was stirred at 0 °C for 10 min until TLC
l
l
4.4. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b- -galactopyranosyl-(13)-2-azido-4,6-O-
benzylidiene-2-deoxy- -glucopyranoside (5)
(n-hexane/EtOAc; 1:1) showed complete consumption of the start-
ing material. After quenching the reaction by adding MeOH (1 mL),
the solvents were evaporated in vacuo and the crude product was
purified by flash chromatography using n-hexane/EtOAc (1:1) to
afford pure disaccharide acceptor 7 (1.6 g, 88%) as white foam.
D
a-D
A mixture of acceptor 3 (1.1 g, 2.75 mmol) and donor 410 (1.8 g,
3.30 mmol) and MS 4Å (2.5 g) in dry CH2Cl2 (25 mL) was stirred
under nitrogen atmosphere for 30 min. Then NIS (970 mg,
4.3 mmol) was added followed by the addition of H2SO4–silica
(100 mg) and the mixture was stirred at 0 °C for 25 min till the
TLC (n-hexane/EtOAc; 1:1) showed complete conversion of the
acceptor 3. The mixture was immediately filtered through a bed
of Celite and washed with CH2Cl2 and the combined filtrate was
washed successively with aq Na2S2O3 (2 ꢃ 25 mL), saturated NaH-
CO3 (2 ꢃ 25 mL), and brine (25 mL). The organic layer was col-
lected, dried over anhydrous Na2SO4, and filtered. The solvents
were evaporated in vacuo. The crude residue was purified by flash
chromatography using n-hexane/EtOAc (1:1.2) to afford pure
½
a 2D5 +98° (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) d: 7.95–6.78
ꢂ
(m, 13H, ArH), 5.95 (dd, 1H, J2 ,3 11.5 Hz, J3 ,4 3.5 Hz, H-30), 5.53
0
0
0
0
(d, 1H, J3 ,4 , 3.5 Hz, H-4 ), 5.49 (d, 1H, J1 ,2 8.5 Hz, H-10), 5.33 (d,
0
0
0
0
0
1H, J1,2 3.5 Hz, H-1), 4.65 (dd, 1H, J1 ,2 8.5 Hz, J2 ,3 11.5 Hz, H-20),
0
0
0
0
0
6a0
4.28 (dd, 1H, J5,
6.5 Hz, J6a ,
12.0 Hz, H-6a0), 4.22(m, 3H, H-
6b0
50, H-6a, H-6b), 4.09 (m, 1H, H-5), 3.93 (dd, 1H, J2,3 11.0 Hz, J3,4
8.5 Hz, H-3), 3.7 (s, 3H, C6H4OCH3), 3.66 (t, 1H, J3,4 J4,5 9.0 Hz, H-
4), 3.17 (dd, 1H, J1,2 3.5 Hz, J2,3 11.0 Hz, H-2), 2.21, 2.07, 1.88 (3s,
9H, 3 ꢃ COCH3). 13C NMR (125 MHz, CDCl3) d: 170.5, 170.1, 169.6
(3 ꢃ COCH3), 168.3, 167.6 (2 ꢃ phthalimido CO), 166.2 (COPh),
134.3, 134.2, 133.0(2), 131.5, 131.3, 130.1, 129.8, 129.7(3), 128.4,
128.3(3), 123.6, 123.5, 118.1(3), 114.5(3) (ArC), 99.9 (C-10), 97.0
(C-1), 55.5 (C6H4OCH3), 20.6, 20.5, 20.4, (3 x COCH3). HRMS calcd
for C40H40N4O16 (M+Na)+: 832.2439, found: 832.2442.
disaccharide 5 (1.9 g, 85%) as white foam. ½a D25
ꢂ
+97° (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3) d: 7.87–6.78 (m, 13H, ArH), 5.86 (dd, 1H,
J2 ,3 11.5 Hz, J3 ,4 3.5 Hz, H-30), 5.57 (s, 1H, CHPh), 5.54 (d, 1H, J1 ,2
0
0
0
0
0
0
8.5 Hz, H-10), 5.44 (d, 1H, J3 ,4 3.5 Hz, H-40), 5.32 (d, 1H, J1,2 3.5 Hz,
0
0
H-1), 4.62 (dd, J1 ,2 8.5 Hz, J2 ,3 11.5 Hz, 1H, H-20), 4.22 (m, 2H, H-3,
H-6a0), 4.12 (dd, 1H, J5,6a 4.0 Hz, J6a,6b 11.0 Hz, H-6a), 4.0 (m, 1H, H-
5), 3.93 (m, 1H, H-50), 3.84 (dd, 1H, J5,6a 5.5 Hz, J6a,6b 11.0 Hz, H-6b),
3.74 (s, 3H, C6H4OCH3), 3.71 (m, 2H, H-4, H-6b0), 3.27 (dd, 1H, J1,2
3.5 Hz, J2,3 9.5 Hz, H-2), 2.17, 1.96, 1.85 (3s, 9H, 3 ꢃ COCH3). 13C
NMR (125 MHz, CDCl3) d: 170.1, 170.0, 169.6 (3 ꢃ COCH3), 168.3,
167.5 (2 ꢃ phthalimido-CO), 136.8, 134.1, 134.0(2), 131.6, 131.5,
131.3, 129.0, 28.1(2), 128.0, 126.1, 126.0(2), 125.8(2), 123.4,
123.3, 118.1, 117.9(2) (ArC), 101.3 (CHPh), 99.2 (C-10), 97.7 (C-1),
55.4 (C6H4OCH3), 20.6, 20.5, 20.4, (3 ꢃ COCH3). HRMS calcd for
0
0
0
0
4.7. p-Tolyl 3,4-di-O-benzyl-2-O-chloroacetyl-1-thio-a-L-
rhamnopyranoside (9)
To a mixture of p-thio-3,4-di-O-benzyl-1-thio-a-L-rhamnopyr-
anoside (8) (3.5 g, 7.8 mmol) in dry CH2Cl2 (20 mL) was added pyr-
idine (5 mL) followed by the addition of choloroacetic anhydride
(2.0 g, 11.7 mmol) keeping the temperature at 0 °C. The reaction
mixture was allowed to stir at the same temperature for 1 h until
complete conversion of the starting material as evident from TLC
(n-hexane/EtOAc; 1:1). Then pyridine was co-evaporated with tol-
uene and the crude product obtained was purified by flash chroma-
tography using n-hexane/EtOAc (2:1) as the eluent to afford pure 9
C
40H40N4O15 (M+Na)+: 816.2490, found: 816.2493.
(3.5 g, 6.65 mmol, 85%) as colorless syrup. ½a D25
ꢂ
+81° (c 1.1, CHCl3).
IR (neat): 3438, 2853, 1721, 1519, 1389, 1267, 1081, 990,
4.5. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b- -galactopyranosyl-(13)-2-azido-2-deoxy-
glucopyranoside (6)
D
a-D-
710 cmꢄ1 1H NMR (500 MHz, CDCl3) d: 7.40–7.15 (m, 14H, ArH),
.
5.68 (dd, 1H, J1,2 2.0 Hz, J2,3 3.5 Hz, H-2), 5.37 (d, J1,2 2.0 Hz, H-1),
4.93 (d, 1H, J 10.5 Hz, CH2Ph), 4.74 (d, 1H, J 11.5 Hz, CH2Ph), 4.65
(d, 1H, J 11.5 Hz, CH2Ph), 4.59 (d, 1H, J 10.5 Hz, CH2Ph), 4.26 (m,
1H, H-5), 4.16 (d, 2H, J 2.5 Hz, COCH2Cl), 3.96 (dd, J2,3 3.5 Hz, J3,4
9.5 Hz, H-3), 3.49 (t, J3,4, J4,5 9.5 Hz, H-4), 2.35 (s, 3H, S-C6H4CH3),
1.36(d, 3H, C–CH3). 13C NMR (125 MHz, CDCl3) d: 138.1(2), 137.4,
132.4(2), 129.9(2), 129.6, 128.6(2), 128.4(2), 128.2(2), 127.9(3),
127.8 (ArC), 86.1 (C-1), 79.9 (C-4), 78.1 (C-3), 72.5 (C-2), 69.0 (C-
5), 40.8 (COCH2Cl), 21.1 (S-C6H4CH3), 17.8 (C–CH3). HRMS calcd
for C29H31ClO5S (M+Na)+: 526.1581, found: 526.1584.
Compound 5 (1.9 g, 2.3 mmol) was dissolved in AcOH/H2O (9:1,
20 mL) and the solution was stirred at 80 °C for 2 h until the start-
ing material was completely converted into a more polar com-
pound as observed by TLC (n-hexane/EtOAc; 1:1). After
evaporating the solvents and co-evaporating with toluene, the
crude product was purified by flash chromatography using n-hex-
ane/EtOAc (1:1) as the eluent to afford pure disaccharide diol 6
(1.6 g, 94%) as white foam. ½a D25
ꢂ
+103° (c 1.0, CHCl3). 1H NMR
0
0
(500 MHz, CDCl3) d: 7.87–6.76 (m, 8H, ArH), 5.92 (dd, 1H, J2 ,3

R. Das, B. Mukhopadhyay / Carbohydrate Research 376 (2013) 1–6
5
4.8. p-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b- -galactopyranosyl-(13)-4-O-(3,4-di-O-benzyl-
-rhamnopyranosyl)-2-azido-6-O-benzoyl-2-deoxy-
glucopyranoside (11)
CH2Cl2 and the combined filtrate was washed successively with aq
Na2S2O3 (2 ꢃ 25 mL), saturated NaHCO3 (2 ꢃ 25 mL), and brine
(25 mL). The organic layer was collected, dried over anhydrous
Na2SO4, and filtered. The solvents were evaporated in vacuo. The
crude residue was purified by flash chromatography using n-hex-
ane/EtOAc (1:1) to afford pure tetrasaccharide 13 as white foam
D
a-
L
a-D-
A mixture of acceptor 7 (1.6 g, 1.9 mmol) and donor 9 (1.3 g,
2.5 mmol) and MS 4Å (2 g) in dry CH2Cl2 (20 mL) was stirred under
nitrogen atmosphere for 30 min. Then NIS (730 mg, 3.25 mmol)
was added followed by the addition of H2SO4–silica (75 mg) and
the mixture was stirred at 0 °C for 20 min till the TLC showed com-
plete consumption of the donor 9. The mixture was immediately
filtered through a bed of Celite and washed with CH2Cl2 and the
combined filtrate was washed successively with aq. Na2S2O3
(2 ꢃ 25 mL), saturated NaHCO3 (2 ꢃ 25 mL), and brine (25 mL).
The organic layer was collected, dried over anhydrous Na2SO4,
and filtered. The solvents were evaporated in vacuo. The crude
product was purified by flash chromatography using n-hexane/
EtOAc (1.5:1) to give the required trisaccharide 10 with traces of
unreacted acceptor 7. The mixture was subjected to the next reac-
tion without further purification. To a solution of the mixture
(1.3 g, 1.05 mmol) in CH2Cl2/MeOH (2:3, 50 mL) was added thio-
urea (400 mg, 5.3 mmol) followed by the addition of 2,4,6-collidine
(1.7 g, 80%). ½a 2D5
ꢂ
+88° (c 0.9, CHCl3). IR (neat): 2940, 1762, 1724,
1515, 1387, 1363, 1239, 1229, 1059, 751, 695 cmꢄ1
.
1H NMR
0
0
(500 MHz, CDCl3) d: 7.97–6.7 (m, 25H, ArH), 6.07 (dd, 1H, J2 ,3
0
11.5 Hz, J3 ,4 3.5 Hz, H-30), 5.49 (d, 1H, J3 ,4 , 3.5 Hz, H-4 ), 5.46 (d,
0
0
0
0
1H, J1 ,2 8.5 Hz, H-10), 5.4 (m, 1H, H-2000), 5.28 (dd, 1H, J3
0
0
000,4000
000,5000
3.5 Hz, J4
10.0 Hz, H-4000), 5.25 (d, 1H, J1,2 3.5 Hz, H-1), 4.98 (m,
2H, H-3000, CH2Ph), 4.94 (s, 1H, H-1000), 4.91 (s, 1H, H-100), 4.8 (m,
2H, H-6a0, CH2Ph), 4.67 (dd, 2H, J 11.5 Hz, CH2Ph), 4.57 (m, 1H,
H-500), 4.51 (dd, 1H, J1 ,2 8.5 Hz, J2 ,3 11.5 Hz, H-20), 4.45 (dd, 1H,
J5, 6a 5.5 Hz, J6a, 6b 10.5 Hz, H-6a), 4.27 (dd, 1H, J5, 6b 8.0 Hz, J6a, 6b
10.5 Hz, H-6b), 4.15 (m, 4H, H-3, H-5, H-50, H-6b0), 4.06 (m, 1H,
H-200), 3.84 (m, 2H, H-300, H-500), 3.75 (t, 1H, J3,4, J4,5 8.5 Hz, H-4),
0
0
0
0
3.72 (s, 3H, C6H4OCH3), 3.61 (t, 1H, J3 ,4 , J4 ,5 9.0 Hz, H-400), 2.95
(dd, 1H, J1,2 3.5 Hz, J2,3 10.0 Hz, H-2), 2.1, 2.05, 1.97, 1.86, 1.82,
1.71 (6s, 8H, 6 ꢃ COCH3), 1.51 (d, 3H, J 6.0 Hz, C–CH3), 1.02 (d,
3H, J 6.5 Hz, C–CH3). 13C NMR (125 MHz, CDCl3) d: 170.5, 170.3,
169.9(2), 169.7, 169.5 (6 ꢃ COCH3), 168.1, 167.6 (2 ꢃ phthalimido
CO), 165.7 (COPh), 138.7, 138.2, 134.3, 134.0, 133.2, 131.9, 129.7,
129.8(2), 129.4, 129.0, 128.5(2), 128.3(3), 128.2(3), 128.1(3),
127.5, 127.3, 127.2(3), 123.5, 123.4, 118.0, 114.7(ArC), 99.7 (C-
100), 99.0 (C-1000), 98.9 (C-10), 96.8(C-1), 55.5 (C6H4OCH3), 22.6,
20.8, 20.7, 20.5, 20.4, 20.0 (6 ꢃ COCH3), 17.6, 17.4 (2 ꢃ C–CH3).
00 00
00 00
(700 lL, 5.3 mmol) and the mixture was allowed to stir under re-
flux for 20 h until complete conversion of the starting material into
a more polar compound as evident from TLC (n-hexane/EtOAc;
1:1). After cooling to room temperature, the solution was diluted
with CH2Cl2 (20 mL) and washed successively with 1 M HCl fol-
lowed by saturated aq. NaHCO3 (2 ꢃ 30 mL) and brine (25 mL).
The organic layer was collected, dried over anhydrous Na2SO4,
and the solvents were evaporated in vacuo. The crude product
was purified by flash chromatography using n-hexane/EtOAc
(1:1) as the eluent to give pure trisaccharide 11 as colorless foam
HRMS calcd for
1430.4858.
C
72H78N4O27 (M+Na)+: 1430.4854, found:
(3.3 g, 78%). ½a 2D5
ꢂ
+121° (c 0.9, CHCl3). IR (neat): 3441, 2847,
4.10. p-Methoxyphenyl 2,3,4-tri-O-acetyl-
rhamnopyranosyl-(12)-3,4-di-O-benzyl-
(14)-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
a-L-
1716, 1513, 1394, 1263, 1088, 995, 703 cmꢄ1
.
1H NMR (500 MHz,
a-L-rhamnopyranosyl-
D-
0
0
0
0
0
CDCl3) d: 8.05–6.63 (m, 24H, ArH), 6.07 (dd, 1H, J2 ,3 11.5 Hz, J3 ,4
0
3.5 Hz, H-30), 5.49 (d, 1H, J3 ,4 , 3.5 Hz, H-4 ), 5.46 (d, 1H, J1 ,2
galactopyranosyl)-2-acetamido-6-O-benzoyl-2-deoxy-a-D-
glucopyranoside (14)
0
0
0
8.5 Hz, H-10), 5.27 (d, 1H, J1,2 3.5 Hz, H-1), 4.98 (d, 1H, J 11.5 Hz,
CH2Ph), 4.93 (s, 1H, H-100), 4.75 (d, 1H, J 11.5 Hz, CH2Ph), 4.67 (m,
3H, CH2Ph, H-6a0), 4.53 (m, 2H, H-20, H-500), 4.42 (dd, 1H, J5,
Compound 13 (1.7 g, 1.2 mmol) was dissolved in thioacetic
acid (20 mL) and the mixture was stirred at room temperature
for 3 days in the dark, until the TLC (n-hexane/EtOAc; 1:1)
showed complete conversion of the starting material. The solvent
was evaporated and co-evaporated with toluene and the crude
residue thus obtained, was purified by flash chromatography to
yield the desired tetrasaccharide 14 (1.2 g, 72%) as white foam.
6a
6.0 Hz, J6a, 6b 11.0 Hz, H-6a), 4.26 (m, 3H, H-3, H-6b, H-6b0), 4.16
(m, 2H, H-5, H-50), 4.12 (m, 1H, H-200), 3.84 (dd, 1H, J2 ,3 3.0 Hz J3 ,4
00 00
00 00
9.0 Hz, H-300), 3.81 (t, 1H, J3,4, J4,5 8.5 Hz, H-4), 3.72 (s, 3H,
C6H4OCH3), 3.52 (t, 1H, J3 ,4 , J4 ,5 9.0 Hz, H-400), 2.95 (dd, 1H, J1,2
3.5 Hz, J2,3 10.0 Hz, H-2), 2.63 (br s, 1H, OH), 2.1, 1.83, 1.71 (3s,
9H, 3 ꢃ COCH3), 1.48 (d, 3H, J 6.5 Hz, C–CH3) 13C NMR (125 MHz,
CDCl3) d: 170.5, 170.3, 169.5 (3 ꢃ COCH3), 168.1, 167.6 (2 ꢃ phtha-
limido CO), 165.9 (COPh), 134.3, 134.0, 133.2, 131.8, 131.2,
129.8(3), 129.5, 128.5(3), 128.4(2), 128.2(3), 127.8(3), 123.6(2),
123.5, 123.4, 118.1(2), 114.6(3),114.5(3) (ArC), 99.5 (C-100), 98.9
(C-10), 97.0 (C-1), 55.5 (C6H4OCH3), 21.0, 20.7, 20.4, (3 ꢃ COCH3),
17.6 (C–CH3). HRMS calcd for C60H62N4O20 (M+Na)+: 1158.3957,
found: 1158.3959.
00 00
00 00
½
a 2D5
ꢂ
+94° (c 0.8, CHCl3). IR (neat): 2935, 1760, 1721, 1511,
1383, 1361, 1249, 1225, 1056, 758, 691 cmꢄ1 1H NMR
.
0
0
(500 MHz, CDCl3) d: 7.97–6.69 (m, 26H, ArH), 5.95 (dd, 1H, J2 ,3
0
12.0 Hz, J3 ,4 3.5 Hz, H-30), 5.78 (d, 1H, J3 ,4 , 3.5 Hz, H-4 ), 5.46
0
0
0
0
(d, 1H, J1 ,2 8.5 Hz, H-10), 5.4 (m, 1H, H-2000), 5.27 (dd, 1H, J3
0
0
000,4000
000,5000
3.5 Hz, J4
10.0 Hz, H-4000), 5.06 (d, 1H, J1,2 3.5 Hz, H-1), 4.95
(m, 3H, H-3000, CH2Ph, H-1000), 4.92 (s, 1H, H-100), 4.71 (m, 4H, H-
6a0, CH2Ph(3)), 4.48 (m, 2H, H-500, H-20), 4.18 (m, 3H, H-6a, H-
6b, H-50), 4.09 (m, 3H, H-3, H-5,, H-6b0), 4.03 (m, 1H, H-200),
3.87 (m, 2H, H-300, H-500), 3.78 (t, 1H, J3,4, J4,5 8.5 Hz, H-4), 3.71
(s, 3H, C6H4OCH3), 3.62 (m, 2H, H-400, H-2), 2.11, 2.06, 2.05,
1.97, 1.90, 1.78, 1.68 (7s, 21H, 7 ꢃ COCH3), 1.56 (d, 3H, J 6.0 Hz,
C–CH3), 1.27 (m, 3H, C–CH3). 13C NMR (125 MHz, CDCl3) d:
170.8, 170.7, 170.6, 170.0, 169.8, 169.7, 169.6 (7 ꢃ COCH3),
168.3, 167.4 (2 ꢃ phthalimido CO), 165.8 (COPh), 134.1, 134.0,
133.11, 131.8, 131.2, 129.8(2), 129.4, 128.4(2), 128.3(3),
128.1(3), 127.9(2), 127.4, 127.3, 127.2(2), 123.5, 123.4, 117.8(2),
114.7(2) (ArC), 99.6 (C-100), 99.0 (C-1000), 98.6 (C-10), 96.6(C-1),
55.5 (C6H4OCH3), 22.6, 20.8, 20.7(2), 20.6, 20.4, 19.9 (7 ꢃ COCH3),
17.9, 17.4 (2 ꢃ C–CH3). HRMS calcd for C69H80N2O28 (M+Na)+:
1384.4898. found: 1384.4899.
4.9. p-Methoxyphenyl 2,3,4-tri-O-acetyl-
(12)-3,4-di-O-benzyl- -rhamnopyranosyl-(14)-3-O-(3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b- -galactopyranosyl)-2-
azido-6-O-benzoyl-2-deoxy- -glucopyranoside (13)
a-L-rhamnopyranosyl-
a-L
D
a
-D
A mixture of acceptor 11 (1.7 g, 1.5 mmol) and donor 12
(750 mg, 1.9 mmol) and MS 4Å (2 g) in dry CH2Cl2 (20 mL) was stir-
red under nitrogen atmosphere for 30 min. Then NIS (555 mg,
2.5 mmol) was added followed by H2SO4–silica (60 mg) and the
mixture was stirred at 0 °C for 20 min till the TLC (n-hexane/EtOAc;
1:1) showed complete consumption of the donor 12. The mixture
was immediately filtered through a bed of Celite and washed with

6
R. Das, B. Mukhopadhyay / Carbohydrate Research 376 (2013) 1–6
4.11. p-Methoxyphenyl a-L-rhamnopyranosyl-(12)-a-L-
Acknowledgement
rhamnopyranosyl-(14)-3-O-(2-acetamido-2-deoxy-b-D-
galactopyranosyl)-2-acetamido-2-deoxy-
(1)
a-D-glucopyranoside
R.D. is thankful to IISER-Kolkata for Junior Research Fellowship.
The work is supported by the research grant 01(2370)/10/EMR-II
from CSIR, New Delhi to B.M.
To the solution of compound 14 (1.1 g, 0.8 mmol) in n-butanol
(20 mL), ethylenediamine (1.2 mL) was added and the reaction
mixture was allowed to stir for 24 h at 110 °C. Then the solvents
were evaporated and co-evaporated with toluene and the crude
product thus obtained is dissolved in pyridine (5 mL) followed by
the addition of Ac2O (5 mL). The reaction mixture was allowed to
stir at room temperature for 10 h when the TLC (n-hexane/EtOAc;
1:1) showed complete conversion of the starting material. The
reaction mixture was then co-evaporated with toluene to give
the required tetrasaccharide, p-methoxyphenyl 2,3,4-tri-O-acetyl-
References
1. Larue, K.; Kimber, M. S.; Ford, R.; Whitfield, C. J. Biol. Chem. 2009, 284, 7395–
7403.
2. Beutin, L.; Wang, Q.; Naumann, D.; Han, W.; Krause, G.; Leomil, L.; Wang, L.;
Feng, L. J. Med. Microbiol. 2007, 56, 177–184.
3. Maciejewska, A.; Lukasiewicz, J.; Niedziela, T.; Szewczuk, Z.; Lugowski, C.
Carbohydr. Res. 2009, 344, 894–900.
4. Pieretti, G.; Corsaro, M. M.; Lanzetta, R.; Parrilli, M.; Canals, R.; Merino, S.;
Momás, J. M. Eur. J. Org. Chem. 2008, 18, 3149–3155.
5. Czaja, J.; Jachymek, W.; Niedziela, T.; Lugowski, C.; Aldova, E.; Kenne, L. Eur. J.
Biochem. 2000, 267, 1672–1679.
6. Niedziela, T.; Dag, S.; Lukasiewicz, J.; Dzieciatkowska, M.; Jachymek, W.;
Lugowski, C.; Kenne, L. Biochemistry 2006, 45, 10422–10433.
7. Säwén, E.; Östervall, J.; Landersjö, C.; Edblad, M.; Weintraub, A.; Ansaruzzaman,
M.; Widmalm, G. Carbohydr. Res. 2012, 348, 99–103.
a
-
L
-rhamnopyranosyl-(12)-3,4-di-O-benzyl-
syl-(14)-3-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
pyranosyl)-2-acetamido-6-O-acetyl-2-deoxy- -glucopyranoside
a
-
L
-rhamnopyrano-
D-galacto-
a
-D
(15). A dilute solution of compound 15 in MeOH (50 mL) and AcOH
(1 mL) was passed through the flow hydrogenation assembly fitted
with a Pd–C cartridge at 50 °C at normal atmospheric pressure of
hydrogen. TLC (CH2Cl2/MeOH; 4:1) showed complete conversion
of the starting material after three cycles. The solvents were evap-
orated in vacuo and the residue was dissolved in MeOH (5 mL) fol-
lowed by the addition of NaOMe in MeOH (1 mL, 0.5 M) and stirred
at 50 °C for 14 h. Then the solution was neutralized by DOWEX
50 W H+ resin. The mixture was filtered through a cotton plug to
remove DOWEX and the filtrate was evaporated in vacuo to afford
pure target tetrasaccharide 1 (330 mg, 80%) as white sticky com-
8. Sau, A.; Panchadhayee, R.; Ghosh, D.; Mishra, A. K. Carbohydr. Res. 2012, 352,
18–22.
9. Verma, P.; Mukhopadhyay, B. Carbohydr. Res. 2009, 344, 2554–2558.
10. Balavoine, G.; Berteina, S.; Gref, A.; Fischer, J. C.; Lubineau, A. J. Carbohydr.
Chem. 1995, 14, 1217–1236.
11. Roy, B.; Field, R. A.; Mukhopadhyay, B. Carbohydr. Res. 2009, 344, 2311–2316.
12. Dasgupta, S.; Mukhopadhyay, B. Eur. J. Org. Chem. 2008, 34, 5770–5777.
13. Roy, B.; Pramanik, K.; Mukhopadhyay, B. Glycoconjugate J. 2008, 25, 157–166.
14. Dasgupta, S.; Pramanik, K.; Mukhopadhyay, B. Tetrahedron 2007, 63, 12310–
12316.
15. Mandal, S.; Mukhopadhyay, B. Tetrahedron 2007, 63, 11363–11370.
16. Medgyes, G.; Jerkovich, G.; Kuszmann, J. Carbohydr. Res. 1989, 186, 225–239.
17. Abbas, S. A.; Haines, A. Carbohydr. Res. 1975, 39, 358–363.
18. Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108, 97–101.
19. Debenham, D. S.; Toone, J. E. Tetrahedron: Asymmetry 2000, 11, 385–387.
20. Rajput, V. K.; Mukhopadhyay, B. J. Org. Chem. 2008, 73, 6924–6927.
21. Bertolini, M. J.; Glaudemans, C. P. J. Carbohydr. Res. 1970, 15, 263–270.
22. Takeo, K.; Maki, K.; Wada, Y.; Kitamura, S. Carbohydr. Res. 1993, 245, 81–96.
23. Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.; Field, R. A. J. Org. Chem. 2004,
69, 7758–7760.
pound. ½a 2D5
ꢂ
+57° (c 0.7, MeOH). IR (neat): 3438, 3019, 1708,
1467, 1233, 773, 672 cmꢄ1
.
1H NMR (500 MHz, D2O) d: 7.04–6.82
(m, 4H, ArH), 5.17 (d, 1H, J1,2 3.5 Hz, H-1), 5.06 (s, 1H, H-1000),
4.92 (d, 1H, J1 ,2 1.0 Hz, H-100), 4.79 (d, 1H, J1 ,2 8.5 Hz, H-10), 4.55
(m, 1H, H-200), 4.16 (m, 2H, H-2, H-2000), 3.97 (m, 1H, H-400), 3.82
(m, 6H, H-3, H-30, H-40, H-4000, H-6a, H-6b), 3.72 (s, 3H, C6H4OCH3),
3.62 (m, 8H, H-4, H-5, H-50, H-500, H-20, H-5000, H-6a0, H-6b0), 3.29 (m,
3H, H-5, H-300, H-3000), 2.07, 1.97 (2s, 6H, 2 ꢃ COCH3), 1.33 (d, 3H, J
6.0 Hz, C–CH3), 1.25 (d, 30H, J 6.5 Hz, C–CH3). 13C NMR (125 MHz,
D2O) d: 174.4, 173.6 (2 ꢃ COCH3), 119.4 (3), 115.6 (2) (ArC), 99.0
(C-1), 99.1 (C-1000), 102.4 (C-10), 104.0 (C-100), 23.3, 23.2 (2 ꢃ COCH3),
18.3, 18.0 (2 ꢃ C–CH3). HRMS calcd for C35H54N2O20 (M+Na)+:
822.3270, found: 822.3273.
00 00
0
0
24. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1996, 292, 71–81.
25. Kanie, O.; Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Carbohydr. Res. 1993, 243,
139–164.
26. Zemplén, G. Ber. Deutch. Chem. Ges. 1926, 59, 1254–1266.
27. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification of Laboratory Chemicals;
Pergamon: London, 1996.