Linear synthesis and conformational analysis of the pentasaccharide repeating unit of the cell wall O-antigen of Escherichia coli O13

Article abstract of DOI:10.1016/j.carres.2014.03.012

Synthesis of the pentasaccharide repeating unit of the O-antigen of Escherichia coli O13 strain has been achieved using a straightforward linear synthetic strategy. Similar reaction conditions have been used for all glycosylations as well as protective group manipulations. All intermediate steps are high yielding and the glycosylation steps are stereoselective. The synthesized pentasaccharide was subjected to conformational analysis using 2D ROESY NMR spectral analysis and molecular dynamics (MD) simulation to get detailed information on conformation of the molecule in aqueous solution.

Full text of DOI:10.1016/j.carres.2014.03.012

Carbohydrate Research 391 (2014) 9–15
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Linear synthesis and conformational analysis of the pentasaccharide
repeating unit of the cell wall O-antigen of Escherichia coli O13
a,
Abhishek Santra a, , Anshupriya Si a, , Rajiv Kumar Kar b, Anirban Bhunia b, , Anup Kumar Misra
⇑
⇑
a Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata 700 054, India
b Bose Institute, Department of Biophysics, P-1/12, C.I.T. Scheme VII-M, Kolkata 700 054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of the pentasaccharide repeating unit of the O-antigen of Escherichia coli O13 strain has been
achieved using a straightforward linear synthetic strategy. Similar reaction conditions have been used
for all glycosylations as well as protective group manipulations. All intermediate steps are high yielding
and the glycosylation steps are stereoselective. The synthesized pentasaccharide was subjected to
conformational analysis using 2D ROESY NMR spectral analysis and molecular dynamics (MD) simulation
to get detailed information on conformation of the molecule in aqueous solution.
Received 23 December 2013
Received in revised form 10 March 2014
Accepted 12 March 2014
Available online 20 March 2014
Keywords:
Pentasaccharide
Ó 2014 Elsevier Ltd. All rights reserved.
O-Antigen
Escherichia coli
Conformational analysis
Molecular dynamics simulation
1. Introduction
also equally immunodominant as the whole polysaccharides.
Therefore, it is quite pertinent to develop glycoconjugate deriva-
Among several bacterial infections in humans, meningitis, and
sepsis,1 diarrhoeal outbreaks2 and urinary tract infections3 are quite
frequent. Pathogenic Escherichia coli (E. coli) strains are mostly
responsible for these infections.4 Although most of the E. coli strains
are usually non-pathogenic members of the human colonic flora,
certain strains acquire virulence factors and contribute to a variety
of infections in humans and animals. E. coli strains are classified
based on three types of antigens, which are (a) somatic (O) antigen;
(b) capsular (K) antigen, and (c) flagellar (H) antigens.5 Recently,
Perepelov et al. reported the structure of the cell wall O-antigen of
tives corresponding to the repeating unit of the cell wall O-anti-
gens for their evaluation in the vaccine development program.9,10
However, isolation of oligosaccharides in adequate quantity avoid-
ing the biological impurities is tedious and somewhat inconve-
nient. Development of chemical synthetic strategies for the
preparation of oligosaccharides as well as their glycoconjugate
derivatives is always beneficial to get significant quantity of pure
compounds. In this context, synthesis of the pentasaccharide
repeating unit of the O-antigen of Escherichia coli O13 as its
p-methoxyphenyl (PMP) glycoside has been undertaken (Fig. 2).
The PMP group has been chosen as a temporary anomeric protect-
ing group which can be removed under an oxidative reaction
condition using ceric ammonium nitrate (CAN) to give the penta-
saccharide hemiacetal derivative. The hemiacetal derivative can
be linked to a protein through a spacer linker to furnish glycocon-
jugate derivatives using methodologies reported elsewhere. Infor-
mation on the conformational orientation of the oligosaccharide in
aqueous environment is the prerequisite parameter for the design-
ing of an effective glycoconjugate derivative for biological studies.
For detailed understanding of the conformational properties of a
molecule, 2D NOESY/ROESY NMR spectral analysis in conjunction
with molecular dynamics (MD) simulation are extremely useful.
A linear synthetic strategy for the synthesis of the pentasaccharide
repeating unit of the O-antigen of E. coli O13 and its detailed
conformational studies in aqueous solution using NMR spectral
analysis and molecular dynamics simulation is presented herein.
E. coli O13 comprised of D-glucose, D-glucosamine, and L-rhamnose
(Fig. 1).6 E. coli O13 strain is associated with diarrheal infections
and shows serological cross-reactivity with Shigella flexneri strains.7
The recent thrust in the drug discovery program is to develop
newer approaches to control bacterial infections due to the emer-
gence of multi-drug resistant bacterial strains.8 Bacterial cell wall
O-antigens are unique polysaccharides and long known for their
association with the bacterial virulence. As a consequence, several
attempts were made in the past toward the development of novel
antibacterial agents or vaccine candidates based on the cell wall
glycoconjugate derivatives.9 Bacterial cell wall O-antigens are
composed of smaller oligosaccharide repeating units, which are
⇑
Corresponding authors. Fax: +91 33 2355 3886 (A.K.M.).
E-mail address: akmisra69@gmail.com (A.K. Misra).
Contributed equally to this work.
http://dx.doi.org/10.1016/j.carres.2014.03.012
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

10
A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
→3)-[α-D-Glcp-(1→2)]-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→2)-α-L-Rhap-(1→2)-α-L-Rhap-
(1→
Figure 1. Structure of the repeating unit of the cell wall O-antigen of Escherichia coli O13.
OPMP
A O
HO
HO
O
OPMP
SEt
SEt
O
O
O
B
BnO
BnO
BnO
HO
HO
HO
BnO
HO
OH
OAc
O
O
3
2
C
O
NHAc
OBn
O
Ph
O
O
D
O
HO
O
BnO
BnO
AcO
SEt
HO
O
OH
OBn
NPhth
5
OH
OH
4
E
O
1
OH
PMP: p-methoxyphenyl
Figure 2. Structure of the synthesized pentasaccharide 1 as its p-methoxyphenyl glycoside and its synthetic intermediates.
1,2-trans glycosylated product (ꢀ10%), which was separated using
column chromatography. Spectral analysis of compound 12
unambiguously supported its formation [signals at d 5.32 (br s,
H-1A), 5.09 (br s, H-1B), 4.92 (br s, H-1D), 4.90 (d, J = 8.5 Hz, H-
1C), 4.75 (br s, H-1E) in the 1H NMR and at d 102.6 (C-1E), 101.2
(C-1B), 97.7 (2 C, C-1A, C-1D), 95.1 (C-1C) in the 13C NMR spectra].
Compound 12 was subjected to a series of reactions involving (a)
transformation of N-phthaloyl group to acetamido group by the
treatment with hydrazine hydrate19 followed by acetylation using
acetic anhydride and pyridine; (b) catalytic transfer hydrogenation
using triethylsilane and 10% Pd–C,20 and (c) saponification using
2. Results and discussion
2.1. Synthesis
The target pentasaccharide 1 was synthesized from the suitably
functionalized monosaccharide intermediates using a sequential
glycosylation strategy using similar reaction conditions for stereo-
selective glycosylations and functional group manipulations. Func-
tionalized monosaccharide derivatives 2,11 3,12 4,13 and 514 were
prepared from the reducing sugars using reaction methodologies
reported earlier (Fig. 2). A general iodonium ion promoted stereo-
selective glycosylation condition was used for the synthesis of
pentasaccharide derivative (12) as well as its synthetic intermedi-
ates 6,15 8, and 10.
sodium methoxide to furnish the pentasaccharide
1 as its
p-methoxyphenyl glycoside in 57% overall yield. Spectral analysis
of compound 1 confirmed its formation [signals at d 5.38 (br s,
H-1A), 5.10 (br s, H-1B), 4.89 (br s, H-1D), 4.76 (d, J = 3.5 Hz, H-
1E), 4.67 (d, J = 8.5 Hz, H-1C) in the 1H NMR and at d 101.9 (C-1C),
101.1 (C-1B), 98.3 (C-1A), 98.1 (C-1D), 97.4 (C-1E) in the 13C NMR
spectra] (Scheme 1).
Stereoselective glycosylation of L-rhamnosyl acceptor 2 and
L
-rhamnosyl donor 3 following the earlier reported reaction condi-
tion15 in the presence of a combination of N-iodosuccinimide (NIS)
and trifluoromethane sulfonic acid (TMSOTf)16,17 furnished disac-
charide derivative 615 in 88% yield. Treatment of compound 6 with
sodium methoxide18 following earlier reported reaction condi-
tions15 gave the disaccharide acceptor 715 in 97% yield. Compound
2.2. Conformational analysis
In order to understand the conformation of the pentasaccharide
1, NOE based 2D NOESY/ROESY NMR experiments in conjunction
with molecular dynamics (MD) simulation were performed. The
NOE cross peaks (interglycosidic linkages) of the pentasaccharide
1 were very weak at 298 K using a 500 MHz spectrometer21 and
therefore, 2D ROESY experiments were carried out to obtain the
structural information of the molecule in aqueous solution. The
2D ROESY spectrum of the pentasaccharide 1 is shown in Figure 3
(and Supporting information), in which all anomeric protons of
compound 1 were clearly identified. The H-1A shows strong ROE
with the aromatic ring protons of –OPMP group (see Supporting
information). A strong ROE was observed between H-1A and H-1B
and medium ROE was observed between H-1A and H-6B, indicating
7 was allowed to couple with
nor 4 in the presence of a combination of NIS-TMSOTf16,17 to fur-
nish trisaccharide derivative in 74% yield. Formation of
D-glucosamine derived glycosyl do-
8
compound 8 was confirmed from its spectral analysis [signals at
d 5.40 (d, J = 8.5 Hz, H-1C), 5.18, (br s, H-1A), 4.91 (br s, H-1B) in
the 1H NMR and at d 101.1 (C-1B), 100.5 (C-1C), 97.5 (C-1A) in the
13C NMR spectra]. Saponification of compound 8 using sodium
methoxide afforded trisaccharide acceptor 9 in 97% yield. Stereose-
lective glycosylation of compound 8 with compound 3 in the pres-
ence of a combination of NIS-TMSOTf16,17 resulted in the formation
of tetrasaccharide derivative 10 in 76% yield, which was confirmed
from its spectral analysis [signals at d 5.24 (d, J = 8.5 Hz, H-1C), 5.18
(br s, H-1A), 4.92 (br s, H-1B), 4.61 (br s, H-1D) in the 1H NMR and at
d 101.1 (C-1C), 100.7 (C-1B), 97.9 (C-1D), 97.6 (C-1A) in the 13C NMR
spectra]. Treatment of compound 10 with sodium methoxide pro-
duced tetrasaccharide acceptor 11 in 94% yield. Stereoselective 1,2-
H-1A is close in proximity to anomeric proton of L-rhamnosyl
moiety B. In addition, few other interglycosidic ROEs such as
H-2A/H-1B, H-3A/H-1B were also visible. The central region
between
L-rhamnosyl moiety B and N-acetyl-D-glucopyranosyl
cis-glycosylation of compound 11 with
D-glucosyl thioglycoside
donor 5 in the presence of a combination of NIS-TMSOTf16,17 in a
mixed solvent (Et2O–CH2Cl2; 4:1, v/v) furnished tetrasaccharide
derivative 12 in 70% yield together with minor quantity of
moiety C has much less conformational freedom since only three
inter-glycosidic ROEs viz. H-2B/H-1C, H-2B/H-2C, and H-1B/H-2C
were observed. The two protons, H-2A and H-2B display the same

A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
11
OPMP
OPMP
A O
BnO
B O
A O
BnO
B O
BnO
BnO
BnO
BnO
a
3
a
4
2 + 3
a
O
O
BnO
BnO
O
OR
Ph
O
O
O
6
7
: R = Ac
C
RO
b
NPhth
: R = H
8
: R = Ac
b
9: R = H
OPMP
OPMP
O
A
O
A
BnO
BnO
BnO
BnO
c, d
O
O
1
5
a
O
B
O
B
BnO
BnO
BnO
Ph
BnO
Ph
O
O
O
O
O
O
O
O
C
C
O
O
NPhth
NPhth
O
D
O
D
BnO
BnO
BnO
BnO
BnO
OR
10
O
PMP: p-methoxyphenyl
BnO
: R = Ac
11: R = H
E
b
BnO
12
O
BnO
Scheme 1. Reagents and conditions: (a) N-iodosuccinimide (NIS), TMSOTf, MS 4 Å, CH2Cl2 (in case of compound 12; CH2Cl2–Et2O, 1:3), À20 °C, 45 min, 88% for compound 6,
74% for compound 8, 76% for compound 10, and 70% for compound 12; (b) 0.1 M CH3ONa, CH3OH, room temperature, 1 h, 97% for compound 7, 97% for compound 9, 94% for
compound 11; (c) (i) NH2NH2ÁH2O, C2H5OH, 80 °C, 8 h; (ii) acetic anhydride, pyridine, room temperature, 2 h; (d) Et3SiH, 10% Pd–C, CH3OH–CH2Cl2 (5:1), room temperature,
8 h; (e) 0.1 M CH3ONa, CH3OH, room temperature, 3 h, 57% over all yield.
Figure 3. 2D ROESY (300 ms mixing time, 500 MHz, 298 K) spectrum of compound 1. The key interresidual cross peaks are marked.
L
-rhamnosyl moiety D and D-glucopyranosyl moiety E of the
chemical shift, and hence it is difficult to distinguish between
H-2A/H-1A and H-2A/H-1B. There were only two inter-glycosidic
ROEs (H-3C/H-1D and H-3C/H-5D) observed between the
pentasaccharide 1 in aqueous solution revealing the distribution
of conformation families at the glycosidic linkages.
The conformational behavior of oligosaccharides in solution
cannot be restricted to a single geometry. Therefore, molecular
N-acetyl-D-glucopyranosyl moiety C and L-rhamnosyl moiety D
and two interglycosidic ROEs (H-2D/H-1E and H-1D/H-1E) between

12
A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
dynamics (MD) simulation has been used as a useful technique
complementary to the 2D ROESY NMR spectral analysis for a de-
tailed understanding on the fluctuations and conformational
changes of a molecule in solution.22 It is noteworthy to mention
that the MD simulation generally performed at a nanosecond
(ns) time scale whereas the NMR window can only take the snaps
conformational analysis, it was found that the compound 1 exists
as a single conformation with a possibility of minor conformational
changes in A–B and B–C portions.
3. Experimental
of conformations at micro to milli second (l
s–ms) time scale.23
3.1. General methods
Therefore, NMR experiments in combination with MD simulation
can provide all types of conformations of a molecule exist in solu-
tion. The proton–proton distances at the interglycosidic linkages of
compound 1 were very good in agreement with the ROE cross
peaks observed in the ROESY spectrum of compound 1 in aqueous
solution (Fig. 4). The terminal two interglycosidic linkages between
D–E moieties and C–D moieties were rigid, as there were almost no
fluctuations of inter proton distances at these linkages. However,
the linkages between the B–C moieties of compound 1 are suffer-
ing from major fluctuations in the interproton distance of H-1B/
H-2C. The inter proton distance between anomeric proton of
All reactions were monitored by thin layer chromatography
over silica gel coated TLC plates. The spots on TLC were visualized
by warming ceric sulfate (2% Ce(SO4)2 in 2 N H2SO4) sprayed plates
in hot plate. Silica gel 230–400 mesh was used for column chroma-
tography. NMR spectra were recorded on Brucker Avance 500 MHz
using CDCl3 as solvent and TMS as internal reference unless stated
otherwise. Chemical shift value is expressed in d ppm. The com-
plete assignment of proton and carbon spectra was carried out
by using a standard set of NMR experiments, for example 1H
NMR, 13C NMR, 13C DEPT 135, 2D COSY, 2D HSQC etc. In addition,
2D ROESY (300 ms mixing time) was performed to assist in the
conformational analysis. The ROESY experiments were performed
with 456 increments in t1 and 2 K data points in t2. The spectral
width was normally 10 ppm in both dimensions. After 16 dummy
scans, 80 scans were recorded per t1 increment. After zero-filling
in t1, 4 K (t2) Â 1 K (t1) data matrices were obtained. The two-
dimensional NMR data were processed by TopSpin software suite
(Bruker, Switzerland). ESI-MS were recorded on a Micromass mass
spectrometer. Optical rotations were recorded in a Jasco P-2000
spectrometer. Commercially available grades of organic solvents
of adequate purity are used in all reactions.
L-rhamnopyranosyl moiety A (H-1A) and methyl protons of
B (H-6B) also showed a huge fluctuation, presumably due to the
fast correlation time of methyl protons (H-6B). The H-1A/H-1B inter
proton distance also fluctuates a small amount within a period of
10 ns simulation. No other significant findings were observed in
the MD simulation of the compound 1. Taken together, this result
indicates that the compound 1 mostly exists in a single conforma-
tion. However, minor conformational changes may also be possible
at A–B and B–C portions of compound 1.
In summary a straightforward synthetic strategy has been
developed for the preparation of the pentasaccharide repeating
unit corresponding to the cell wall O-antigen of Escherichia coli
O13. A linear glycosylation approach has been adopted for the
construction of the target pentasaccharide as its p-methoxyphenyl
glycoside. All glycosylation steps gave good yields with excellent
stereoselectivity. The conformational analysis of the pentasaccha-
ride 1 in aqueous solution was carried out using 2D ROESY NMR
spectral analysis and MD simulation techniques. From the
3.1.1. p-Methoxyphenyl (2-O-acetyl-3,4-di-O-benzyl-
a-L-rhamno-
pyranosyl)-(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (6)
a-L
Prepared by the glycosylation of compound 2 and compound 3
in 88% yield following the similar reaction condition reported
earlier.15
Figure 4. (A) The intermolecular ROEs of pentasaccharide 1. (B) The global minimum conformation of pentasaccharide 1, according to the molecular modeling technique.

A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
13
3.1.2. p-Methoxyphenyl (3,4-di-O-benzyl-
a
-
L
-rhamnopyranosyl)-
J = 9.5 Hz each, 1H, H-4A), 3.11 (t, J = 9.5 Hz each, 1H, H-4B), 1.18–
1.17 (m, 6H, 2 CCH3); 13C NMR (125 MHz, CDCl3): d 154.8–113.7
(Ar-C), 101.9 (PhCH), 101.2 (C-1C), 97.6 (C-1A), 82.0 (C-4C), 80.5
(C-4A), 80.4 (C-4B), 79.3 (C-2A), 78.9 (C-3A), 77.7 (C-3B), 75.6 (C-
2B), 75.3 (PhCH2), 75.0 (PhCH2), 72.6 (PhCH2), 71.9 (PhCH2), 68.6
(C-3C), 68.5 (C-6C), 68.3 (C-5A), 68.1 (C-5B), 65.7 (C-5C), 56.6 (C-
2C), 55.5 (OCH3), 18.0, 17.7 (2CCH3); ESI-MS: 1178.4 [M+Na]+; Anal.
Calcd for C68H69NO16 (1155.46): C, 70.63; H, 6.01; found: C, 70.50;
H, 6.15.
(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (7)
a
-L
Prepared from compound 6 in 97% yield following the similar
reaction condition reported earlier.15
3.1.3. p-Methoxyphenyl (3-O-acetyl-4,6-O-benzylidene-2-deoxy-
2-N-phthalimido-b-
-rhamnopyranosyl)-(1?2)-3,4-di-O-benzyl-
anoside (8)
D
-glucopyranosyl)-(1?2)-(3,4-di-O-benzyl-
a
-
L
a-L
-rhamnopyr-
A solution of compound 7 (1.5 g, 1.93 mmol), compound 4 (1 g,
2.07 mmol), and MS-4 Å (1 g) in anhydrous CH2Cl2 (10 mL) was
stirred under argon at room temperature for 30 min. The reaction
mixture was cooled to À20 °C and N-iodosuccinimide (NIS;
500 mg, 2.22 mmol) and trimethylsilyl trifluoromethanesulfonate
3.1.5. p-Methoxyphenyl (2-O-acetyl-3,4-di-O-benzyl-
pyranosyl)-(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-
b- -glucopyranosyl)-(1?2)-(3,4-di-O-benzyl- -rhamnopyrano-
syl)-(1?2)-3,4-di-O-benzyl- -rhamnopyranoside (10)
a-L-rhamno-
D
a-L
a-L
(TMSOTf; 5
l
L) were added to it and stirred at same temperature
A solution of compound 9 (1.2 g, 1.04 mmol), compound 3
(500 mg, 1.16 mmol), and MS-4 Å (1 g) in anhydrous CH2Cl2
(10 mL) was stirred under argon at room temperature for 30 min.
The reaction mixture was cooled to À20 °C and NIS (280 mg,
for 45 min. The reaction mixture was filtered through a CeliteÒ
bed and washed with CH2Cl2 (100 mL). The combined organic layer
was successively washed with 5% Na2S2O3, satd. NaHCO3 and
water, dried (Na2SO4), and concentrated. The crude product was
purified over SiO2 using hexane–EtOAc (6:1) as eluant to give pure
1.24 mmol) and TMSOTf (3 lL) were added to it and stirred at same
temperature for 45 min. The reaction mixture was filtered through
a CeliteÒ bed and washed with CH2Cl2 (100 mL). The combined or-
ganic layer was successively washed with 5% Na2S2O3, satd.
NaHCO3 and water, dried (Na2SO4), and concentrated. The crude
product was purified over SiO2 using hexane–EtOAc (6:1) as eluant
to give pure compound 10 (1.2 g, 76%). White solid; mp 175–
compound 8 (1.7 g, 74%). White solid; mp 177–178 °C; [
a
]
D
À55.7
(c 1.0, CHCl3); IR (KBr): 3031, 2925, 2861, 1775, 1745, 1718, 1507,
1389, 1223, 1079, 1049, 722, 697 cmÀ1; 1H NMR (500 MHz, CDCl3):
d 7.88–7.07 (m, 29H, Ar-H), 6.85 (d, J = 9.0 Hz, 2H, Ar-H), 6.76 (d,
J = 9.0 Hz, 2H, Ar-H), 6.05 (t, J = 10.0 Hz each, 1H, H-3C), 5.45 (s,
1H, PhCH), 5.40 (d, J = 8.5 Hz, 1H, H-1C), 5.18, (br s, 1H, H-1A),
4.91 (br s, 1H, H-1B), 4.78 (d, J = 11.0 Hz, 1H, PhCH2), 4.66 (br s,
2H, PhCH2), 4.54 (d, J = 11.0 Hz, 1H, PhCH2), 4.39 (t, J = 10.0 Hz
each, 1H, H-2C), 4.31 (d, J = 11.0 Hz, 1H, PhCH2), 4.08 (2 d,
J = 11.0 Hz each, 2H, PhCH2), 3.96–3.94 (m, 2H, H-2A, PhCH2),
3.90 (2 dd, J = 9.5 Hz, 3.0 Hz, 2H, H-3A, H-3B), 3.74 (s, 3H, OCH3),
3.72–3.65 (m, 3H, H-2B, H-6abC), 3.64–3.59 (m, 2H, H-5A, H-5B),
3.56 (t, J = 10.0 Hz each, 1H, H-4C), 3.54–3.50 (m, 1H, H-5C), 3.19
(t, J = 9.5 Hz each, 1H, H-4A), 3.48 (t, J = 9.5 Hz each, 1H, H-4B),
176 °C; [
1715, 1507, 1454, 1388, 1232, 1103, 1075, 1051, 987, 723,
697 cmÀ1 1H NMR (500 MHz, CDCl3): d 5.46 (s, 1H, PhCH), 5.24
a]
À35 (c 1.0, CHCl3); IR (KBr): 3030, 2932, 1774, 1744,
D
;
(d, J = 8.5 Hz, 1H, H-1C), 5.18 (br s, 1H, H-1A), 4.92 (br s, 1H, H-
1B), 4.89–4.88 (m, 1H, H-2D), 4.80 (2d, J = 11.0 Hz each, 2H, PhCH2),
4.74 (t, J = 9.5 Hz each, 1H, H-3C), 4.66 (br s, 2H, PhCH2), 4.61 (br s,
1H, H-1D), 4.54–4.46 (m, 3H, PhCH2), 4.41 (d, J = 11.0 Hz, 1H,
PhCH2), 4.36 (t, J = 9.5 Hz each, 1H, H-2C), 4.31 (d, J = 11.0 Hz, 1H,
PhCH2), 4.09–4.06 (m, 2H, PhCH2), 3.95–3.92 (m, 2H, H-2A, PhCH2),
3.90–3.86 (m, 3H, H-3D, H-5A, H-5B), 3.82 (dd, J = 10.0, 3.0 Hz, 1H,
H-3A), 3.74 (s, 3H, OCH3), 3.71–3.69 (m, 2H, H-2B, H-3B), 3.65–
3.52 (m, 4H, H-4C, H-5D, H-6abC), 3.45–3.39 (m, 1H, H-5C), 3.23 (t,
J = 9.5 Hz each, 1H, H-4A), 3.17 (t, J = 9.5 Hz each, 1H, H-4B), 3.08
(t, J = 9.5 Hz each, 1H, H-4D), 1.82 (s, 3H, COCH3), 1.18–1.16 (m,
6H, 2 CCH3), 0.80 (d, J = 6.0 Hz, 3H, CCH3); 13C NMR (125 MHz,
CDCl3): d 169.6 (COCH3), 154.7–114.5 (Ar-C), 101.7 (PhCH), 101.1
(C-1C), 100.7 (C-1B), 97.9 (C-1D), 97.6 (C-1A), 80.5 (C-4D), 80.4 (C-
4B), 80.2 (C-4A), 80.0 (C-4C), 79.1 (C-3D), 78.9 (C-3A), 77.7 (C-2A),
76.9 (C-3B), 75.5 (C-2B), 75.3 (PhCH2), 75.0 (PhCH2), 74.6 (C-3C),
72.6 (PhCH2), 71.9 (PhCH2), 71.4 (PhCH2), 69.1 (C-2D), 68.6 (C-6C),
68.5 (C-5D), 68.3 (C-5A), 67.8 (C-5B), 66.1 (C-5C), 56.5 (C-2C), 55.5
(OCH3), 20.7 (COCH3), 18.1 (CCH3), 17.7 (CCH3), 17.3 (CCH3); MAL-
DI-MS: 1546.5 [M+Na]+; Anal. Calcd for C90H93NO21 (1523.62): C,
70.90; H, 6.15; found: C, 70.76; H, 6.30.
1.95 (s, 3H, COCH3), 1.18–1.15 (m, 6H,
2
CCH3); 13C NMR
(125 MHz, CDCl3): d 169.9 (COCH3), 156.2–114.5 (Ar-C), 101.6
(PhCH), 101.1 (C-1B), 100.5 (C-1C), 97.5 (C-1A), 80.5 (2 C, C-4A, C-
4B), 79.2 (2 C, C-3A, C-3B), 78.9 (C-2A), 77.7 (C-4C), 75.6 (C-2B),
75.3 (PhCH2), 75.0 (PhCH2), 72.7 (PhCH2), 71.9 (PhCH2), 69.3 (C-
3C), 68.6 (C-6C), 68.4 (C-5A), 68.3 (C-5B), 65.7 (C-5C), 55.5 (OCH3),
55.3 (C-2C), 20.7 (COCH3), 18.1, 17.7 (2 CCH3); ESI-MS: 1220.4
[M+Na]+; Anal. Calcd for C70H71NO17 (1197.47): C, 70.16; H, 5.97;
found: C, 70.00; H, 6.10.
3.1.4. p-Methoxyphenyl (4,6-O-benzylidene-2-deoxy-2-N-phtha
limido-b-D-glucopyranosyl)-(1?2)-(3,4-di-O-benzyl-
a-L-rhamno-
-rhamnopyranoside (9)
pyranosyl)-(1?2)-3,4-di-O-benzyl-
a-L
A solution of compound 8 (1.5 g, 1.25 mmol) in 0.1 M CH3ONa
in CH3OH (50 mL) was allowed to stir at room temperature for
1 h. The reaction mixture was neutralized with Dowex 50 W-X8
(H+) resin, filtered, and concentrated. The crude product was
passed through a small pad of SiO2 using hexane–EtOAc (1:1) as
eluant to give pure compound 9 (1.4 g, 97%). White solid; mp
3.1.6. p-Methoxyphenyl (3,4-di-O-benzyl-
(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-b-
pyranosyl)-(1?2)-(3,4-di-O-benzyl- -rhamnopyranosyl)-(1?2)-
3,4-di-O-benzyl- -rhamnopyranoside (11)
a-L
-rhamnopyranosyl)-
D-gluco-
a-L
84–85 °C; [
a
]
À51 (c 1.0, CHCl3); IR (KBr): 2928, 1715, 1507,
a-L
D
1390, 1077, 1050, 697 cmÀ1
;
1H NMR (500 MHz, CDCl3): d 7.59–
A solution of compound 10 (1.1 g, 0.72 mmol) in 0.1 M CH3ONa
in CH3OH (40 mL) was allowed to stir at room temperature for 1 h.
The reaction mixture was neutralized with Dowex 50 W-X8 (H+)
resin, filtered, and concentrated. The crude product was passed
through a small pad of SiO2 using hexane–EtOAc (1:1) as eluant
to give pure compound 11 (1 g, 94%). White solid; mp 165–
7.08 (m, 29H, Ar-H), 6.85 (d, J = 9.0 Hz, 2H, Ar-H), 6.76 (d,
J = 9.0 Hz, 2H, Ar-H), 5.49 (s, 1H, PhCH), 5.25 (d, J = 8.5 Hz, 1H, H-
1C), 5.18 (br s, 1H, H-1A), 4.93 (br s, 1H, H-1B), 4.81 (t, J = 9.5 Hz
each, 1H, H-3C), 4.76 (d, J = 11.0 Hz, 1H, PhCH2), 4.66 (br s, 2H,
PhCH2), 4.53 (d, J = 11.0 Hz, 1 H PhCH2), 4.36 (t, J = 9.5 Hz each,
1H, H-2C), 4.28 (d, J = 11.0 Hz, 1H, PhCH2), 4.12 (d, J = 11.0 Hz, 1H,
PhCH2), 4.06 (d, J = 11.0 Hz, 1H, PhCH2), 3.97 (d, J = 11.0 Hz, 1H,
PhCH2), 3.95 (br s, 1H, H-2B), 3.92–3.88 (m, 2H, H-3B, H-6aC), 3.78
(dd, J = 9.5 Hz, 3.0 Hz, 1H, H-3A), 3.74 (s, 3H, OCH3), 3.72 (br s,
1H, H-2A), 3.71–3.69 (m, 1H, H-5A), 3.68–3.65 (m, 1H, H-5B),
3.64–3.53 (m, 3H, H-4C, H-6bC), 3.43–3.38 (m, 1H, H-5C), 3.17 (t,
166 °C; [
a
]
À49 (c 1.0, CHCl3); IR (KBr): 3036, 2939, 1741, 1517,
D
1455, 1230, 1077, 1056, 977, 699 cmÀ1; 1H NMR (500 MHz, CDCl3):
d 7.61–7.09 (m, 39H, Ar-H), 6.86 (d, J = 9.0 Hz, 2H, Ar-H), 6.77 (d,
J = 9.0 Hz, 2H, Ar-H), 5.46 (s, 1H, PhCH), 5.26 (d, J = 8.5 Hz, 1H, H-
1C), 5.19 (br s, 1H, H-1A), 4.92 (br s, 1H, H-1B), 4.82 (t, J = 9.5 Hz
each, 1H, H-1C), 4.79–4.74 (m, 2H, PhCH2), 4.68 (br s, 1H, H-1D),

14
A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
4.67 (br s, 2H, PhCH2), 4.57 (br s, 2H, PhCH2), 4.55–4.48 (m, 2H,
PhCH2), 4.37 (t, J = 9.0 Hz each, 1H, H-2C), 4.31 (d, J = 11.0 Hz, 1H,
PhCH2), 4.08–4.05 (m, 2H, PhCH2), 3.95–3.88 (m, 5H, H-2A, H-3A,
H-3D, H-5A, H-5B), 3.76–3.75 (m, 2H, H-2D, H-3B), 3.74 (s, 3H,
OCH3), 3.71–3.70 (m, 1H, H-2B), 3.68–3.53 (m,4H, H-4C, H-5D, H-
removed under reduced pressure and the crude product was dis-
solved in acetic anhydride and pyridine (5 mL, 1:1 v/v) and kept
at room temperature for 2 h and concentrated under reduced pres-
sure. To a solution of the acetylated product and 10% Pd–C
(150 mg) in CH3OH–CH2Cl2 (10 mL; 5:1, v/v) was added Et3SiH
(1 mL, 6.26 mmol) drop wise during 30 min and the reaction mix-
ture was allowed to stir for 8 h. The reaction mixture was filtered
through a CeliteÒ bed, washed with CH3OH (50 mL), and concen-
trated under reduced pressure. A solution of the hydrogenolyzed
product in 0.1 M CH3ONa in CH3OH (15 mL) was allowed to stir
at room temperature for 3 h. The reaction mixture was neutralized
using Dowex 50W X8 (H+) resin, filtered, and concentrated
under reduced pressure to give compound 1, which was passed
through column of Sephadex LH-20 gel using CH3OH–H2O (3:1)
as eluant to give pure compound 1 as p-methoxyphenyl glycoside
6abC), 3.48–3.43 (m, 1H, H-5C), 3.23 (t, J = 10.5 Hz each, 1H, H-4A),
3.22 (t, J = 10.5 Hz each, 1H, H-4B), 3.07 (t, J = 10.5 Hz each, 1H,
H-4D), 1.20–1.17 (m, 6H, 2 CCH3), 0.81 (d, J = 6.0 Hz, 3H, CCH3);
13C NMR (125 MHz, CDCl3): d 154.8–114.5 (Ar-C), 101.8 (PhCH),
101.1 (C-1C), 100.6 (C-1B), 99.2 (C-1D), 97.6 (C-1A), 80.6 (C-4D),
80.5 (C-4B), 80.4 (C-4A), 80.0 (C-4C), 79.6 (C-3D), 79.0 (C-3A), 78.9
(C-2A), 77.7 (C-3B), 75.5 (C-2B), 75.3 (PhCH2), 75.0 (2 C, 2 PhCH2),
73.3 (C-3C), 72.7 (PhCH2), 71.9 (2 C, 2 PhCH2), 68.6 (2 C, C-2D, C-
5D), 68.5 (C-6C), 68.3 (C-5A), 67.6 (C-5B), 65.9 (C-5C), 56.8 (C-2C),
55.5 (OCH3), 18.1 (CCH3), 17.3 (CCH3); MALDI-MS: 1504.6
[M+Na]+; Anal. Calcd for C88H91NO20 (1481.6): C, 71.29; H, 6.19;
found: C, 71.15; H, 6.36.
(160 mg, 57%). White powder; [
a]
D
À0.1 (c 1.0, H2O); IR (KBr):
3434, 2946, 1629, 1362, 1152, 667 cmÀ1
;
1H NMR (500 MHz,
D2O): d 6.99 (d, J = 9.0 Hz, 2H, Ar-H), 6.88 (d, J = 9.0 Hz, 2H, Ar-
H), 5.38 (br s, 1H, H-1A), 5.10 (br s, 1H, H-1B), 4.89 (br s, 1H, H-
1D), 4.76 (d, J = 3.5 Hz, 1H, H-1E), 4.67 (d, J = 8.5 Hz, 1H, H-1C),
4.07–4.06 (m, 2H, H-2A, H-2B), 3.97 (dd, J = 10.0, 3.5 Hz, 1H, H-
3A), 3.94–3.85 (m, 2H, H-3C, H-5D), 3.83–3.73 (m, 5H, H-2D, H-3B,
H-3D, H-4C, H-6aC), 3.71–3.69 (m, 3H, H-2C, H-6abE), 3.70 (s, 3H,
OCH3), 3.66–3.61 (m, 3H, H-5A, H-5E, H-6bC), 3.54 (t, J = 10.0 Hz
each, 1H, H-4E), 3.44–3.40 (m, 4H, H-2E, H-3E, H-5B, H-5C), 3.36
(t, J = 9.5 Hz each, 2H, H-4A, H-4D), 3.24 (t, J = 9.5 Hz each, 1H, H-
4B), 1.97 (s, 3H, COCH3), 1.15–1.12 (m, 9H, 3 CCH3); 13C NMR
(125 MHz, D2O): d 174.6 (COCH3), 154.9–115.2 (Ar-C), 101.9 (C-
1C), 101.1 (C-1B), 98.3 (C-1A), 98.1 (C-1D), 97.4 (C-1E), 81.2 (C-4E),
78.8 (C-2A), 78.7 (C-2B), 76.2 (C-2D), 75.8 (C-3C), 72.7 (C-5C), 72.1
(C-3D), 72.0 (C-3E), 71.8 (C-2E), 71.7 (C-4B), 71.3 (C-4A), 69.7 (C-
3A), 69.6 (2 C, C-5A, C-5B), 69.4 (C-5E), 69.3 (2 C, C-3B, C-4D), 69.2
(C-5D), 68.2 (C-4C), 60.6 (C-6C), 60.1 (C-6E), 55.8 (2 C, C-2C,
OCH3), 22.3 (COCH3), 16.6, 16.5, 16.3 (3 CCH3); ESI-MS: 950.3
[M+Na]+; Anal. Calcd for C39H61NO24 (927.35): C, 50.48; H, 6.63;
found: C, 50.31; H, 6.82.
3.1.7. p-Methoxyphenyl (2,3,4,6-tetra-O-benzyl-
osyl)-(1?2)-(3,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-(4,6-
O-benzylidene-2-deoxy-2-N-phthalimido-b- -glucopyranosyl)-
(1?2)-(3,4-di-O-benzyl- -rhamnopyranosyl)-(1?2)-3,4-di-O
-benzyl- -rhamnopyranoside (12)
a-D-glucopyran-
a-L
D
a
-L
a
-L
To a solution of compound 11 (800 mg, 0.54 mmol) and com-
pound 5 (340 mg, 0.58 mmol) in anhydrous CH2Cl2–Et2O (10 mL;
1:3, v/v) was added MS-4 Å (1 g) and the reaction mixture was stir-
red under argon at room temperature for 30 min. The reaction mix-
ture was cooled to À20 °C. To the cooled reaction mixture were
added NIS (140 mg, 0.62 mmol) and TMSOTf (2 lL) and it was stir-
red at same temperature for 45 min. The reaction mixture was fil-
tered through a CeliteÒ bed and washed with CH2Cl2 (100 mL). The
combined organic layer was successively washed with 5% Na2S2O3,
satd. NaHCO3 and water, dried (Na2SO4), and concentrated. The
crude product was purified over SiO2 using hexane–EtOAc (3:1)
as eluant to give pure compound 12 (760 mg, 70%). Colorless oil;
[a
]
D
À2 (c 1.0, CHCl3); IR (neat): 3030, 2931, 1678, 1507, 1454,
1364, 1214, 1086, 1050, 735, 696 cmÀ1; 1H NMR (500 MHz, CDCl3):
d 7.49–7.12 (m, 59H, Ar-H), 6.92 (d, J = 9.0 Hz, 2H, Ar-H), 6.81 (d,
J = 9.0 Hz, 2H, Ar-H), 5.44 (br s, 1H, PhCH), 5.32 (br s, 1H, H-1A),
5.09 (br s, 1H, H-1B), 4.92 (br s, 1H, H-1D), 4.90 (d, J = 8.5 Hz, 1H,
H-1C), 4.81 (t, J = 10.0 Hz each, 1H, H-3C), 4.80–4.76 (m, 2H, PhCH2),
4.75 (br s, 1H, H-1E), 4.73–4.60 (m, 15H, PhCH2), 4.50–4.45 (m, 3H,
H-2C, PhCH2), 4.33 (d, J = 11.0 Hz, 1H, PhCH2), 4.10–3.95 (m, 6H, H-
2A, H-2B, H-2D, H-3A, H-5A, H-5B), 3.92–3.89 (m, 2H, H-3B, H-4C),
3.85–3.79 (m, 2H, H-3D, H-5D), 3.77 (s, 3H, OCH3), 3.65–3.58 (m,
3H, H-2E, H-3E, H-4A), 3.55–3.50 (m, 3H, H-4E, H-5C, H-6aE), 3.47–
3.39 (m, 5H, H-4B, H-4D, H-6abC, H-6bE), 3.36–3.30 (m, 1H, H-5E),
1.70 (s, 3H, COCH3), 1.31–1.28 (m, 6H, 2 CCH3), 0.95 (d, J = 6.0 Hz,
3H, CCH3); 13C NMR (125 MHz, CDCl3): d 156.2–114.6 (Ar-C),
102.6 (C-1E), 101.6 (PhCH), 101.2 (C-1B), 97.7 (2 C, C-1A, C-1D),
95.1 (C-1C), 81.6 (C-4B), 80.9 (C-4D), 80.3 (2 C, C-4A, C-4C), 79.8
(C-3D), 79.2 (2 C, C-3A, C-3B), 78.7 (C-4E), 77.7 (C-3E), 77.6 (C-2B),
75.6 (PhCH2), 75.5 (PhCH2), 75.4 (2 C, C-2E, PhCH2), 75.3 (2 C, C-
2A, PhCH2), 74.9 (PhCH2), 73.4 (2 C, 2 PhCH2), 72.9 (2 C, C-5B,
PhCH2), 72.2 (PhCH2), 71.8 (PhCH2), 70.2 (C-3C), 68.6 (2 C, C-6C,
C-6E), 68.5 (2 C, C-2D, C-5D), 68.4 (C-5A), 68.3 (C-5C), 66.2 (C-5E),
57.5 (C-2C), 55.5 (OCH3), 18.3, 18.2, 17.9 (3 CCH3); MALDI-MS:
2026.8 [M+Na]+; Anal. Calcd for C122H125NO25 (2003.85): C,
73.07; H, 6.28; found: C, 72.90; H, 6.50.
3.2. Molecular dynamics simulation
The model structure of the pentasaccharide 1 was prepared
using GLYCAM_06j force field.24 The non-electrostatic model of
p-methoxyphenoxy group (OPMP) was prepared and the param-
eters were adopted using general AMBER force field (GAFF).25
For molecular dynamics simulation, the pentasaccharide 1 was
solvated in truncated octahedral box with TIP326 water models
with an edge distance of 8 Å. Accounts of the nonbonded interac-
tions were done with a cutoff distance of 8 Å and simulation was
performed under isothermal-isobaric periodic boundary condi-
tions. SHAKE algorithm was applied to restrain all hydrogen
bonds with an integration time step of 2 fs. Minimization of the
system, was performed with 1500 steepest descent steps fol-
lowed by 500 conjugate gradient steps with convergence criteria
of 0.01 kcal molÀ1. The system annealed for a time period of
50 ps, and it was stabilized at 300 K. Density equilibrium of the
system was processed for another 50 ps before the production
runs with weak restraints over the solute atoms. Finally the ex-
plicit simulation was carried out for a time scale of 10 ns using
sander module of AMBER11.
Acknowledgements
3.1.8. p-Methoxyphenyl (
nopyranosyl)-(1?3)-(2-acetamido-2-deoxy-b-
(1?2)-( -rhamnopyranosyl)-(1?2)- -rhamnopyranoside (1)
To a solution of compound 12 (600 mg, 0.3 mmol) in C2H5OH
(20 mL) was added hydrazine monohydrate (0.3 mL) and the
reaction was allowed to stir at 80 °C for 8 h. The solvents were
a
-
D
-glucopyranosyl)-(1?2)-(
a-L-rham-
A.S. and A.S. thank CSIR, India for providing a Senior and Junior
Research Fellowships, respectively. This work was supported by
DST, India (Project No. SR/S1/OC-83/2010). Mr. Barun Mazumdar,
Bose Institute NMR facility is thankfully acknowledged for carrying
out the NMR experiments with great care.
D-glucopyranosyl)-
a-L
a-L

A. Santra et al. / Carbohydrate Research 391 (2014) 9–15
11. Guchhait, G.; Misra, A. K. Tetrahedron: Asymmetry 2009, 20, 1791–1797.
15
Supplementary data
12. Sajtos, F.; Hajkó, J.; Kövér, K. E.; Lipták, A. Carbohydr. Res. 2001, 334,
253–259.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.03.
012.
13. Kihlberg, J. O.; Leigh, D. A.; Bundle, D. R. J. Org. Chem. 1990, 55, 2860–2863.
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17. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31,
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