Convergent synthesis of a common pentasaccharide corresponding to the O-antigen of Escherichia coli O168 and Shigella dysenteriae type 4

Article abstract of DOI:10.1007/s10719-010-9317-y

A convenient synthetic strategy of the common acidic pentasaccharide repeating unit corresponding to the O-antigen of enterotoxigenic E. coli O168 and Shigella dysenteriae type 4 has been successfully developed. A stereoselective [2+3] block glycosylation method has been exploited to get the target pentasaccharide derivative. Most of the synthetic intermediates were solid and prepared in high yields from commercially available reducing sugars following a series of protection-deprotection reactions. A α-D-mannose moiety has been used as the source of α-D-glucosamine moiety. A late-stage TEMPO mediated selective oxidation reaction finally resulted in the pentasaccharide containing a glucuronic acid unit.

Full text of DOI:10.1007/s10719-010-9317-y

Glycoconj J (2011) 28:11–19
DOI 10.1007/s10719-010-9317-y
Convergent synthesis of a common pentasaccharide
corresponding to the O-antigen of Escherichia coli O168
and Shigella dysenteriae type 4
Goutam Guchhait & Anup Kumar Misra
Received: 25 September 2010 /Revised: 29 November 2010 /Accepted: 2 December 2010 /Published online: 22 December 2010
#
Springer Science+Business Media, LLC 2010
Abstract A convenient synthetic strategy of the common
acidic pentasaccharide repeating unit corresponding to the O-
antigen of enterotoxigenic E. coli O168 and Shigella
dysenteriae type 4 has been successfully developed. A
stereoselective [2+3] block glycosylation method has been
exploited to get the target pentasaccharide derivative. Most
of the synthetic intermediates were solid and prepared in
high yields from commercially available reducing sugars
following a series of protection-deprotection reactions. A α-
D-mannose moiety has been used as the source of α-D-
glucosamine moiety. A late-stage TEMPO mediated selec-
tive oxidation reaction finally resulted in the pentasaccharide
containing a glucuronic acid unit.
vitamins [1], a number of virulent E. coli strains are
responsible for severe enteric/diarrhoeal infections [2, 3]. In
general, the enteropathogenic strain of E. coli have been
classified in several classes [4, 5], which include (a)
enteropathogenic E. coli (EPEC), (b) enteroinvasive E. coli
(EIEC), (c) enterotoxigenic E. coli (ETEC), (d) enter-
oaggregative E. coli (EAEC), (e) diffusely adherent E. coli
(DAEC), and (f) enterohemorrhagic E. coli (EHEC) etc.
ETEC strains are the major cause of traveller’s diarrhoea.
The pathogen adheres to the mucosa in the small intestine
and produce toxins that act on mucosal cells to cause
diarrhoea. In general, two types of toxins are produced by
ETEC strains, heat-labile (LT) and heat-stable (ST) [6, 7].
ETEC colonizes the surface of the small bowel mucosa and
elaborates enterotoxins, which give rise to intestinal
secretion.
.
.
Keywords Lipopolysaccharides Glycosylations
.
.
Antigens Escherichia coli Shigella dysenteriae
Similar to ETEC, Shigella dysenteriae is the most
virulent among the pathogenic bacilli of the genus Shigella
[8]. They are responsible for severe intestinal diseases
including dysentery and have the potential for causing
disastrous health problems in developing countries [9, 10].
Virulent E. coli and Shigella strains have long been known
to be closely related in terms of their gene sequences and
pathogenic actions [11, 12]. Recently, Perepelove et al.
reported [13] the structure of a common oligosaccharide
repeating unit corresponding to the O-polysaccharide of
enterotoxigenic E. coli O168 and revised structure of the O-
antigen of Shigella dysenteriae type 4 (Fig. 1). Earlier
Dmitriev et al. [14] reported a structure of the O-antigen of
Shigella dysenteriae type 4, which was partially incorrect
(Fig. 2). Both strains are highly associated with several
diarrhoeal pandemic.
Introduction
Diarrhoea is a common infectious disease in infants and
children in tropical countries and in places where sanitary
conditions are poor. The major causative agents for the
diarrhoeal infections are enteropathogenic Escherichia coli
(E. coli) strains. Although, E. coli serves a useful function
in human body by suppressing the growth of harmful
bacteria and by synthesising considerable amounts of
Electronic supplementary material The online version of this article
(doi:10.1007/s10719-010-9317-y) contains supplementary material,
which is available to authorized users.
:
G. Guchhait A. K. Misra (*)
Molecular Medicine Division, Bose Institute,
P-1/12, C. I. T. Scheme VII M,
Kolkata 700054, India
Although, several therapeutics have appeared to control
these infections, emergance of the drug resistant strains
necessitates the development of alternative approaches for
e-mail: akmisra69@gmail.com

12
Glycoconj J (2011) 28:11–19
Fig. 1 Common pentasaccharide repeating unit of the O-antigen of E. coli O168 and Shigella dysenteriae type 4 (revised structure) [13]
controlling these infections [15]. Since, O-polysaccharide is
highly immunogenic and important virulence factor, earlier
several attempts have been made to develop O-polysaccha-
ride derived glycoconjugates for their use as antibacterial
vaccine candidates [16–19]. For a detailed understanding of
the pathogenic role of the O-antigens, several biological
experiments are necessary demanding large quantities of
oligosaccharides in hand. Although, oligosaccharides can
be isolated from the natural source, concise chemical
synthesis only provide the access to a large quantity of a
particular oligosaccharide. Since, both enterotoxigenic E.
coli O168 and Shigella dysenteriae type 4 have common
structure of their O-antigens as well as their pathogenic
actions, it would be beneficial to synthesize this oligosac-
charide for its use in the development of glycoconjugate for
biological evaluation as anti-diarrhaeal agents. Although, a
synthesis of the incorrect structure of the O-antigen of
Shigella dysenteriae type 4 has been reported earlier [20],
development of a synthetic strategy for the revised structure
is quite essential to validate biological function of the O-
antigen. In this context, we report herein convenient
synthesis of a common pentasaccharide as its 4-
methoxyphenyl glycoside (1) corresponding to the O-
antigen of enterotoxigenic E. coli O168 and revised
structure of Shigella dysenteriae type 4 using sequential
and [2+3] block glycosylation strategies (Fig. 3). 4-
Methoxyphenyl (PMP) group can be easily removed under
oxidative condition to provide the pentasaccharide hemiacetal
for its further use.
1,2-trans linked α-D-mannosidic moiety as the precursor of
1,2-cis linked α-D-glucosamine unit in pentasaccharide
derivative 12, (b) late stage TEMPO mediated selective
oxidation of primary hydroxyl group to the carboxylic
functionality, (c) stereoselective 1,2-cis glycosylation using
L-fucose derivatives, (d) use of 4-methoxyphenyl group as
the anomeric protection for its easy removal under
oxidative condition. In order to construct the target
pentasaccharide (1), a series of suitably protected mono-
saccharide intermediates 2 [21], 3 [22] and 4 were prepared
from the commercially available reducing sugars using
literature reported protection-deprotection methodologies.
Ethyl (2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1→3)-2-O-
acetyl-4,6-O-benzylidene-1-thio-α-D-mannopyranoside (5)
was synthesized from L-fucose derivative and D-mannose
derivatives through a stereoselective “armed-disarmed”
glycosylation following the method reported earlier [20]
(Fig. 3).
Ethyl 2,3,6-tri-O-benzyl-1-thio-β-D-glucopyranoside (6)
[23] was allowed to react with 4-methoxybenzyl chloride in
the presence of sodium hydroxide [24] to give ethyl 2,3,6-
tri-O-benzyl-4-O-(4-methoxybenzyl)-1-thio-β-D-glucopyr-
anoside (4) in 89% yield. Stereoselective 1,2-cis-glycosyl-
ation of compound 2 with L-fucose derived thioglycoside
derivative (3) in the presence of N-iodosuccinimide (NIS)
and trimethylsilyl trifluoromethanesulfonate (TMSOTf)
[25, 26] furnished disaccharide derivative 7 in 85% yield.
Appearance of characteristic signal in the NMR spectra [δ
1
4.82 (d, J=3.4 Hz, H-1B) in the H NMR and δ 99.5 (JC-1/
H-1=171 Hz, C-1B) in the 13C NMR spectra] confirmed its
stereoselective formation. Compound 7 was subjected to a
sequence of reactions involving saponification using sodi-
um methoxide and selective acetylation via orthoesterifica-
tion [27] using triethyl orthoacetate in the presence of p-
toluenesulfonic acid followed by hydrolysis to give
disaccharide derivative 8 in 90% yield. NMR spectra of
compound 8 supported its formation. Stereoselective 1,2-cis
glycosylation of compound 8 with thioglycoside derivative
4 in the presence of NIS-TMSOTf [25, 26] furnished
Results and discussion
The synthesis of the target pentasaccharide poses a number
of challenges because of the presence of several 1,2-cis
glycosidic linkages between the monosaccharide units. The
final compound has been synthesized applying sequential
and block glycosylation strategies. Following features can
be found in this convenient synthetic strategy: (a) use of
Fig. 2 Incorrect structure of the pentasaccharide repeating unit of the O-antigen of Shigella dysenteriae type 4 reported earlier (Dmitriev et al.) [14]

Glycoconj J (2011) 28:11–19
13
OH
1
4.81–4.79 (m, H-2D) in H NMR and δ 97.8 (C-1D) in the
13C NMR spectra]. Compound 12 was subjected to a set of
reactions involving (a) treatment with ethylene diamine
followed by N-acetylation to transform N-phthalimido
group to acetamido group [30], (b) selective hydrogenolysis
[31] to remove benzyl ethers and reduction of azido group
to amine keeping benzylidene acetals unaffected followed
by N-acetylation using conventional acetylation and de-O-
acetylation, (c) selective TEMPO mediated oxidation [32–
34] of the primary hydroxyl group to the carboxylic group
under a phase-transfer condition and finally (d) removal of
benzylidene acetals under hydrogenolysis [31] to furnish
target pentasaccharide (1) as its 4-methoxyphenyl glycoside
in 57% over all yield. Spectral analysis of compound 1
confirmed its formation [signals at δ 5.18 (d, J=3.7 Hz, H-
1B), 5.16 (br s, H-1D), 5.15 (br s, H-1E), 5.05 (d, J=3.4 Hz,
O
HO
A
OPMP
NHAc
O
B
O
OH
O
HO
HO
HO
C
O
ONa
O
AcHN
O
D
O
O
OH
HO
OH
PMP: 4-Methoxyphenyl
E O
OH
HO
1
SEt
Ph
O
O
O
O
HO
OBn
OPMP
NPhth
OAc
AcO
1
2
3
H-1C), 4.69 (br s, H-1A) in the H NMR and δ 100.5 (2 C,
OAc
Ph
O
C-1D, C-1E), 100.1 (C-1A), 99.9 (C-1B), 99.5 (C-1C) in the
O
13C NMR spectra] (Fig. 4, Scheme 1).
OBn
O
O
O
MBnO
BnO
SEt
SEt
O
OBn
OBn
OBn
4
BnO
5
Conclusion
In conclusion, a convergent chemical synthesis of a common
pentasaccharide repeating unit of the O-antigen of E. coli
O168 and Shigella dysenteriea type 4 as its 4-methoxyphenyl
glycoside has been successfully developed using a block
synthetic strategy. Most of the intermediates are solid and all
glycosylation steps are highly stereoselective and reproduc-
ible for scale-up preparation. A α-D-mannosyl moiety has
been used as the source of α-D-glucosaminyl moiety to get
exclusively 1,2-cis linked α-D-glucosaminyl unit in the
pentasaccharide. Selective TEMPO mediated oxidation of a
primary hydroxyl group in a pentasaccharide derivative was
achieved using a two-step, one-pot phase transfer oxidation
protocol without affecting other secondary hydroxyl groups
present in the molecules. 4-Methoxyphenyl group has been
chosen as the temporary protecting group at the reducing end.
Fig. 3 Structure of the synthesized pentasaccharide (1) as its 4-
methoxyphenyl glycoside corresponding to the common O-antigen of
E. coli O168 and Shigella dysenteriae type 4
trisaccharide derivative 9 in 81% yield together with minor
quantities of 1,2-trans glycosylated product (~10%), which
was confirmed from its spectral analysis [appearance of
characteristic signal at δ 4.83 (d, J=3.6 Hz, H-1C) in the 1H
NMR and δ 99.0 (JC-1/H-1=172 Hz, C-1C) in the 13C NMR
spectra]. Oxidative removal [28] of 4-methoxybenzyl group
from compound 9 using 2,3-dichloro-5,6-dicyano-p-benzo-
quinone (DDQ) afforded trisaccharide acceptor 10 in 75%
yield. Stereoselective block glycosylation of compound 10
with disaccharide thioglycoside 5 in the presence of NIS-
TMSOTf [25, 26] furnished pentasaccharide derivative 11
in 80% yield. Appearance of new characteristic signals at δ
5.34 (br s, H-1D), 4.94 (d, J=2.8 Hz, H-1E) in the 1H NMR
and δ 99.8 (JC-1/H-1=174 Hz, C-1D), 94.0 (JC-1/H-1=172 Hz,
C-1E) in the 13C NMR spectra of compound 11 supported
its formation. The 2-O-acetyl group of the D-mannosyl
moiety in compound 11 was chemoselectively removed
using dilute sodium methoxide keeping the 4-O-acetyl
group in the L-fucosyl moiety intact exploiting the
difference of reactivity of two O-acetyl groups. The
selective deacetylated product was allowed to react with
trifluoromethanesulfonic anhydride in the presence of
pyridine and the resulting triflate derivative was treated
with sodium azide to furnish compound 12 in 76% over all
yield [29]. Formation of compound 12 was confirmed from
its spectral analysis [signals at δ 5.44 (br s, 1 H, H-1D),
Experimental section
General methods All reactions were monitored by thin
layer chromatography over silica gel coated TLC plates.
The spots on TLC were visualized by warming ceric
sulphate (2% Ce(SO4)2 in 2N H2SO4) sprayed plates in
hot plate. Silica gel 230-400 mesh was used for column
chromatography. 1H and 13C NMR, 2D COSY, HMQC
spectra were recorded on Brucker Avance DRX 500 MHz
using CDCl3 and D2O as solvents and TMS as internal
reference unless stated otherwise. Chemical shift value is
expressed in δ ppm. ESI-MS were recorded on a Micro-
mass Quttro mass spectrometer. Elementary analysis was

14
Glycoconj J (2011) 28:11–19
Fig. 4 1H NMR spectrum of
synthesized pentasaccharide
as its 4-methoxyphenyl glycoside
(1)
carried out on Carlo Erba-1108 analyzer. Optical rotations
were measured at 25°C on a Jasco P-2000 polarimeter.
Commercially available grades of organic solvents of
adequate purity are used in all reactions.
4.04 mmol) in dry DMF (10 mL) were added powdered
NaOH (0.5 g, 12.5 mmol) and 4-methoxybenzyl chloride
(1.0 mL, 7.37 mmol) and the reaction mixture was allowed
to stir at room temperature for 3 h. The reaction mixture was
poured into water and extracted with CH2Cl2 (100 mL). The
organic layer was washed with water, dried (Na2SO4) and
concentrated under reduced pressure. The crude product was
Ethyl 2,3,6-tri-O-benzyl-4-O-(4-methoxybenzyl)-1-thio-β-
D-glucopyranoside (4) To a solution of compound 6 (2.0 g,
OBn
O
OBn
O
MBnO
a
HO
SEt
SEt
BnO
BnO
OBn
OBn
6
4
Ph
O
Ph
O
b
O
4
O
O
O
2 + 3
A
A
B O
O
OPMP
O
OPMP
b
AcO
BnO
NPhth
NPhth
O
B
O
OBn
OBn
9:
BnO
C
OR2
RO
1
R = MBn
O
OBn
R O
e
7: R1 = R2 = Ac
10: R = H
c, d
1
2
8
: R = Ac; R = H
Ph
O
O
O
A
O
OPMP
h, i, j, k, l
5
b
1
NPhth
B
O
OBn
R1
O
Ph
O
O
O
AcO
BnO
D
O
R2
C
BnO
f, g
O
OBn
E
O
O
OBn
OBn
BnO
1
2
11:
R = OAc; R = H
12: R1 = H; R2 = N3
Scheme 1 Reagents: (a) 4-Methoxybenzyl chloride, NaOH, DMF,
room temperature, 3 h, 89%; (b) N-iodosuccinimide (NIS), TMSOTf,
CH2Cl2, −30°C, 1 h, 85% for 7, 81% for 9 and 80% for 11; (c) 0.05 M
CH3ONa, CH3OH, room temperature, 1 h; (d) (i) triethylorthoacetate, p-
TsOH, DMF, room temperature, 1 h; (ii) 80% AcOH, room temperature,
30 min, 90% in two steps; (e) DDQ, CH2Cl2, H2O, room temperature, 2
h, 75%; (f) 0.05 M CH3ONa, CH3OH, room temperature, 2 h; (g) (i)
triflic anhydride, pyridine, CH2Cl2, 0°C, 8 h; (ii) NaN3, DMF, 70°C, 3 h,
76% in two steps; (h) (i) ethylene diamine, n-BuOH, 90°C, 6 h; (ii)
acetic anhydride, pyridine, room temperature, 2 h; (i) H2, 20% Pd(OH)2-
C, CH3OH-EtOAc (1:1 v/v), room temperature, 6 h; (j) (i) acetic
anhydride, pyridine, room temperature, 2 h; (ii) 0.1 M CH3ONa,
CH3OH, room temperature, 5 h; (k) (i) NaBr, CH2Cl2, H2O, TBAB,
TEMPO, NaHCO3, NaOCl, 0–5°C, 3 h; (ii) tert-butanol, 2-methyl-but-2-
ene, NaClO2, NaH2PO4, room temperature, 3 h; (l) H2, 20% Pd(OH)2-C,
CH3OH, room temperature, 24 h, 57% in seven steps

Glycoconj J (2011) 28:11–19
15
purified over SiO2 using hexane-EtOAc (5:1) as eluant to give
pure 4 (2.2 g, 89%) as white solid. m.p. 82°C; [α]D25 −5.2 (c
1.6, CHCl3); IR (KBr): 3436, 3061, 3030, 2954, 2905, 2788,
1962, 1759, 1614, 1587, 1515, 1496, 1452, 1401, 1362,
(s, 3 H, COCH3), 1.69 (s, 3 H, COCH3), 0.54 (d, J=6.4 Hz,
3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 170.8, 170.1
(2 COCH3), 168.2 (2 C, Phth), 156.0–114.9 (Ar-C), 102.5
(PhCH), 99.5 (JC-1/H-1=171 Hz; C-1B), 98.7 (JC-1/H-1
=
1
1250, 1083, 990, 950, 737, 699 cm−1; H NMR (500 MHz,
163.2 Hz; C-1A), 81.6 (C-5A), 75.6 (C-3A), 73.2 (C-2B),
73.1 (PhCH2), 72.1 (C-4B), 70.8 (C-3B), 69.1 (C-6A), 67.0
(C-4A), 65.5 (C-5B), 56.1 (C-2A), 56.0 (OCH3), 20.9 (2 C,
COCH3), 15.5 (CCH3); ESI-MS: 846.2 [M+Na]+; Anal.
Calcd. for C45H45NO14 (823.28): C, 65.61; H, 5.51%;
found: C, 65.39; H, 5.76%.
CDCl3): δ 7.25–7.18 (m, 15 H, Ar-H), 6.97 (d, J=8.6 Hz,
2 H, Ar-H), 6.68 (d, J=8.6 Hz, 2 H, Ar-H), 4.82 (2 d, J=
11.0 Hz, 2 H, PhCH2), 4.76 (d, J=11.0 Hz, 1 H, PhCH2),
4.63 (2 d, J=11.0 Hz, 2 H, PhCH2), 4.51 (d, J=12.0 Hz, 1 H,
PhCH2), 4.45 (d, J=12.0 Hz, 1 H, PhCH2), 4.40 (d, J=
10.5 Hz, 1 H, PhCH2), 4.35 (d, J=9.7 Hz, 1 H, H-1), 3.67 (s,
3 H, OCH3), 3.64 (dd, J=10.9, 1.8 Hz, 1 H, H-6a), 3.58–3.53
(m, 2 H, H-3, H-6b), 3.49 (t, J=9.4 Hz, 1 H, H-4), 3.35–3.32
(m, 2 H, H-2, H-5), 2.69–2.64 (m, 2 H, SCH2CH3), 1.25
(t, J=7.4 Hz, 3 H, SCH2CH3); 13C NMR (125 MHz, CDCl3):
δ 159.7–114.2 (Ar-C), 87.1 (C-1), 85.5 (C-5), 82.2 (C-2), 79.5
(C-3), 78.0 (C-4), 76.0 (PhCH2), 75.8 (PhCH2), 75.0
(PhCH2), 73.8 (PhCH2), 69.5 (C-6), 55.6 (OCH3), 25.3
(SCH2CH3), 15.6 (SCH2CH3); ESI-MS: 637.2 [M+Na]+;
Anal. Calcd. for C37H42O6S (614.27): C, 72.28; H, 6.89%;
found: C, 72.10; H, 7.12%.
4-Methoxyphenyl (4-O-acetyl-2-O-benzyl-α-L-fucopyrano-
syl)-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-
D-glucopyranoside (8) A solution of compound 7 (2.7 g,
3.28 mmol) in 0.05 M CH3ONa in CH3OH (50 mL) was
allowed to stir at room temperature for 1 h and neutralized
with Dowex-50W X8 (H+) resin. The reaction mixture was
filtered and concentrated under reduced pressure. To a
solution of the crude product in dry DMF (20 mL) was
added triethylorthoacetate (3.0 mL, 16.36 mmol) followed by
p-TsOH (250 mg) and the reaction mixture was allowed to stir
at room temperatute for 1 h. The solvents were removed
under reduced pressure and a solution of the crude orthoester
derivative in 80% aq. AcOH (50 mL) was allowed to stir at
room temperature for 30 min. The reaction mixture was
evaporated and co-evaporated with toluene (3×100 mL) to
give the crude product, which was purified over SiO2 using
hexane-EtOAc (5:1) as eluant to give pure 8 (2.3 g, 90%) as
4-Methoxyphenyl (3,4-di-O-acetyl-2-O-benzyl-α-L-fucopyr-
anosyl)-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthali-
mido-β-D-glucopyranoside (7) To a solution of compound
2 (2.0 g, 3.97 mmol) and compound 3 (1.8 g, 4.70 mmol) in
anhydrous CH2Cl2 (20 mL) was added MS 4Å (5 g) and the
reaction mixture was allowed to stir at room temperature for
1 h under argon. The reaction mixture was cooled to −30°C
and N-iodosuccinimide (NIS; 1.3 g, 5.77 mmol) followed
by trimethylsilyltrifluoromethane sulfonate (TMSOTf;
25 μL) were added to it and the reaction mixture was
allowed to stir at same temperature for 1 h. The reaction
mixture was quenched with Et3N (0.1 mL), filtered through
a Celite® bed and washed with CH2Cl2 (100 mL). The
organic layer was washed with 5% aq. Na2S2O3, satd. aq.
NaHCO3 and water, dried (Na2SO4) and concentrated to
dryness. The crude product was purified over SiO2 using
hexane-EtOAc (6:1) as eluant to give pure 7 (2.8 g, 85%) as
25
white solid. m.p. 81°C; [α]D −26.7 (c 1.6, CHCl3); IR
(KBr): 3480, 3061, 3023, 2961, 2932, 1778, 1738, 1714,
1507, 1455, 1390, 1243, 1222, 1099, 1031, 997, 965, 754,
722, 699 cm−1; 1H NMR (500 MHz, CDCl3): δ 7.57–7.13 (m,
14 H, Ar-H), 6.74 (d, J=9.0 Hz, 2 H, Ar-H), 6.64 (d, J=
9.1 Hz, 2 H, Ar-H), 5.70 (d, J=8.5 Hz, 1 H, H-1A), 5.48 (s,
1 H, PhCH), 4.93–4.92 (m, 1 H, H-4B), 4.78 (d, J=3.3 Hz, H-
1B), 4.64 (dd, J=8.5, 8.5 Hz, 1 H, H-3A), 4.49 (dd, J=8.5,
8.5 Hz, 1 H, H-2A), 4.36–4.33 (m, 1 H, H-6aA), 4.17–4.12 (m,
2 H, H-5B, PhCH2), 3.87 (dd, J=10.0, 3.5 Hz, 1 H, H-3B),
3.82 (d, J=12.5 Hz, 1 H, PhCH2), 3.79–3.65 (m, 2 H, H-4A,
H-5A), 3.63 (s, 3 H, OCH3), 3.27 (dd, J=10.0, 3.3 Hz, 1 H,
H-2B), 1.90 (s, 3 H, COCH3), 0.63 (d, J=6.5 Hz, 3H, CCH3);
13C NMR (125 MHz, CDCl3): δ 171.2 (COCH3), 168.8 (2 C,
Phth), 156.0–114.9 (Ar-C), 102.1 (PhCH), 98.8 (C-1B), 98.6
(C-1A), 81.9 (C-5A), 76.0 (C-2B), 75.5 (C-3A), 73.9 (C-4B),
72.7 (PhCH2), 69.0 (C-6A), 68.4 (C-3B), 66.9 (C-4A), 65.9 (C-
5B), 56.2 (C-2A), 55.9 (OCH3), 21.1 (COCH3), 16.0 (CCH3);
ESI-MS: 804.2 [M+Na]+; Anal. Calcd. for C43H43NO13
(781.27): C, 66.06; H, 5.54%; found: C, 65.83; H, 5.80%.
25
white solid. m.p. 79°C; [α]D −26.7 (c 1.6, CHCl3); IR
(KBr): 3476, 3064, 3033, 2981, 2932, 2873, 1778, 1738,
1714, 1507, 1455, 1390, 1243, 1222, 1099, 1031, 997, 965,
755, 722, 699, cm−1; 1H NMR (500 MHz, CDCl3): δ 7.68–
7.19 (m, 14 H, Ar-H), 6.84 (d, J=9.1 Hz, 2 H, Ar-H), 6.72
(d, J=9.0 Hz, 2 H, Ar-H), 5.75 (d, J=8.5 Hz, 1 H, H-1A),
5.57 (s, 1 H, PhCH), 5.12 (dd, J=10.6, 3.2 Hz, 1 H, H-3B),
5.07–5.06 (m, 1 H, H-4B), 4.82 (d, J=3.4 Hz, 1 H, H-1B),
4.78 (dd, J=8.8, 8.8 Hz, 1 H, H-3A), 4.61 (dd, J=8.5,
8.5 Hz, 1 H, H-2A), 4.43–4.40 (m, 1 H, H-6aA), 4.28–4.24
(m, 1 H, H-5B), 4.06 (d, J=12.9 Hz, 1 H, PhCH2), 3.94 (d,
J=12.9 Hz, 1 H, PhCH2), 3.90–3.86 (m, 1 H, H-66A), 3.82
(t, J=9.1 Hz, 1 H, H-4A), 3.79–3.74 (m, 1 H, H-5A), 3.70
(s, 3 H, OCH3), 3.52 (dd, J=10.5, 3.5 Hz, 1 H, H-2B), 1.93
4-Methoxyphenyl [2,3,6-tri-O-benzyl-4-O-(4-methoxyben-
zyl)-α-D-glucopyranosyl]-(1→3)-(4-O-acetyl-2-O-benzyl-
α-L-fucopyranosyl)-(1→3)-4,6-O-benzylidene-2-deoxy-2-
N-phthalimido-β-D-glucopyranoside (9) To a solution of

16
Glycoconj J (2011) 28:11–19
compound 8 (2.1 g, 2.68 mmol) and compound 4 (2.0 g,
3.25 mmol) in anhydrous CH2Cl2 (20 mL) was added MS
4Å (4 g) and the reaction mixture was allowed to stir at
room temperature for 1 h under argon. The reaction mixture
was cooled to −30°C and NIS (0.9 g, 4.0 mmol) followed
by TMSOTf (10 μL) were added to it and the reaction
mixture was allowed to stir at same temperature for 1 h.
The reaction mixture was quenched with Et3N (0.1 mL),
filtered through a Celite® bed and washed with CH2Cl2
(100 mL). The organic layer was washed with 5% aq.
Na2S2O3, satd. aq. NaHCO3 and water, dried (Na2SO4) and
concentrated to dryness. The crude product was purified over
SiO2 using hexane-EtOAc (5:1) as eluant to give pure 9
(2.9 g, 81%) as white solid. m.p. 86°C; [α]D25 +43.7 (c 1.6,
CHCl3); IR (KBr): 3450, 3063, 3031, 2932, 2869, 1778,
1741, 1715, 1507, 1455, 1390, 1371, 1222, 1100, 1028, 969,
The crude product was purified over SiO2 using hexane-
EtOAc (4:1) as eluant to give pure 10 (1.7 gmg, 75%) as
25
white solid. m.p. 84°C; [α]D +40.2 (c 1.6, CHCl3); IR
(KBr): 3476, 3087, 3063, 3032, 2933, 2870, 1778, 1740,
1715, 1507, 1454, 1390, 1372, 1222, 1099, 1058, 1027,
969, 965, 829, 755, 738, 722, 698 cm−1; 1H NMR
(500 MHz, CDCl3): δ 7.38–7.13 (m, 29 H, Ar-H), 6.82
(d, J=9.0 Hz, 2 H, Ar-H), 6.71 (d, J=9.1 Hz, 2 H, Ar-H),
5.76 (d, J=8.5 Hz, 1 H, H-1A), 5.48 (s, 1 H, PhCH), 5.05
(br s, 1 H, H-4B), 4.85 (d, J=11.4 Hz, 1 H, PhCH2), 4.80
(d, J=3.3 Hz, 1 H, H-1C), 4.73 (d, J=11.4 Hz, 1 H,
PhCH2), 4.67 (d, J=3.3 Hz, 1 H, H-1B), 4.64–4.63 (m, 4 H,
PhCH2), 4.57 (dd, J=8.5, 8.5 Hz, 1 H, H-2A), 4.44–4.35
(m, 3 H, H-3A, PhCH2), 4.25 (d, J=12.9 Hz, 1 H, H-6aA),
4.15–4.13 (m, 1 H, H-5B), 4.10 (dd, J=10.0, 3.5 Hz, 1 H,
H-3B), 3.85–3.72 (m, 6 H, H-3C, H-4C, H-5A, H-6bA, H-
1
965, 755, 722, 697 cm−1; H NMR (500 MHz, CDCl3): δ
6abC), 3.68 (s, 3 H, OCH3), 3.59–3.58 (m, 2 H, H-4A, H-
7.36–7.05 (m, 33 H, Ar-H), 6.78 (d, J=9.0 Hz, 2 H, Ar-H),
6.73 (d, J=9.1 Hz, 2 H, Ar-H), 5.75 (d, J=8.5 Hz, 1 H, H-
1A), 5.51 (s, 1 H, PhCH), 5.05 (br s, 1 H, H-4B), 4.83 (d, J=
3.6 Hz, 1 H, H-1C), 4.80 (d, J=11.8 Hz, 1 H, PhCH2), 4.75
(d, J=11.8 Hz, 1 H, PhCH2), 4.67 (d, J=3.4 Hz, 1 H, H-1B),
4.70–4.64 (m, 4 H, H-3A, PhCH2), 4.57 (dd, J=8.5, 8.5 Hz,
1 H, H-2A), 4.50–4.39 (m, 6 H, H-6aA, PhCH2), 4.30 (d, J=
12.9 Hz, 1 H, H-6aC), 4.15–4.13 (m, 1 H, H-5B), 4.07 (dd, J=
10.0, 3.5 Hz, 1 H, H-3B), 3.86–3.78 (m, 1 H, H-6bA), 3.75 (s,
3 H, OCH3), 3.70 (s, 3 H, OCH3), 3.78–3.68 (m, 5 H, H-3C,
H-4A, H-5A, H-5C, H-6bC), 3.59 (t, J=9.1 Hz, 1 H, H-4C),
3.50 (dd, J=10.2, 3.3 Hz, 1 H, H-2B), 3.35 (dd, J=9.8,
3.6 Hz, 1 H, H-2C), 1.99 (s, 3 H, COCH3), 0.58 (d, J=
6.5 Hz, 3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 170.8
(COCH3), 168.7 (2 C, Phth), 159.4–114.0 (Ar-C), 102.2
5C), 3.50 (dd, J=10.2, 3.3 Hz, 1 H, H-2B), 3.32 (dd, J=9.8,
3.6 Hz, 1 H, H-2C), 2.00 (s, 3 H, COCH3), 0.62 (d, J=
6.5 Hz, 3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 171.1
(COCH3), 167.9 (2 C, Phth), 156.0–114.9 (Ar-C), 102.2
(PhCH), 99.8 (C-1B), 98.7 (C-1C), 98.6 (C-1A), 81.9 (C-
5A), 81.3 (C-5C), 79.6 (C-2C), 76.1 (C-2B), 75.4 (PhCH2),
75.0 (C-4C), 74.5 (PhCH2), 73.7 (C-4B), 72.9, 72.8 (2
PhCH2), 71.8 (C-3B), 71.7 (C-3C), 70.1 (C-6C), 69.1 (C-
6A), 67.0 (C-4A), 66.1 (C-5B), 56.1 (C-2A), 55.9 (OCH3),
21.3 (COCH3), 16.1 (CCH3); ESI-MS: 1236.4 [M+Na]+;
Anal. Calcd. for C70H71NO18 (1213.46): C, 69.24; H,
5.89%; found: C, 69.00; H, 6.15%.
4-Methoxyphenyl (2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-
(1→3)-(2-O-acetyl-4,6-O-benzylidene-α-D-mannopyranosyl)-
(1→4)-(2,3,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(4-O-
acetyl-2-O-benzyl-α-L-fucopyranosyl)-(1→3)-4,6-O-benzyli-
dene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (11) To
a solution of compound 10 (1.5 g, 1.23 mmol) and
compound 5 (1.1 g, 1.42 mmol) in anhydrous CH2Cl2
(15 mL) was added MS 4Å (3 g) and the reaction mixture
was allowed to stir at room temperature for 1 h under
argon. The reaction mixture was cooled to −30°C and NIS
(380 mg, 1.68 mmol) followed by TMSOTf (5 μL) were
added to it and the reaction mixture was allowed to stir at
same temperature for 1 h. The reaction mixture was
quenched with Et3N (0.1 mL), filtered through a Celite®
bed and washed with CH2Cl2 (100 mL). The organic layer
was washed with 5% aq. Na2S2O3, satd. aq. NaHCO3 and
water, dried (Na2SO4) and concentrated to dryness. The
crude product was purified over SiO2 using hexane-EtOAc
(5:1) as eluant to give pure 11 (1.9 g, 80%) as white solid;
(PhCH), 99.9 (JC-1/H-1=171 Hz; C-1B), 99.0 (JC-1/H-1
=
172 Hz; C-1C), 98.6 (JC-1/H-1=164 Hz; C-1A), 81.9 (C-5A),
81.8 (C-5C), 80.0 (C-2C), 77.7 (C-4C), 76.0 (C-3A), 75.7
(PhCH2), 75.0 (C-2B), 74.6 (PhCH2), 74.5 (C-4B), 74.4 (C-
3C), 73.8 (PhCH2), 72.89, 72.82 (2 PhCH2), 71.4 (C-3B),
69.1 (C-6A), 69.0 (C-6C), 66.9 (C-4A), 66.3 (C-5B), 56.1 (C-
2A), 55.9, 55.5 (2 OCH3), 21.3 (COCH3), 16.1 (CCH3); ESI-
MS: 1356.5 [M+Na]+; Anal. Calcd. for C78H79NO19
(1333.52): C, 70.20; H, 5.97%; found: C, 70.00; H, 6.22%.
4-Methoxyphenyl (2,3,6-tri-O-benzyl-α-D-glucopyranosyl)-
(1→3)-(4-O-acetyl-2-O-benzyl-α-L-fucopyranosyl)-(1→3)-4,6-
O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside
(10) To a solution of 9 (2.5 g, 1.87 mmol) in CH2Cl2
(30 mL) was added a solution of 2,3-dicholoro-5,6-
dicyano-p-benzoquinone (DDQ, 850 mg, 3.74 mmol) in
H2O (15 mL) and the biphasic reaction mixture was
allowed to stir at room temperature for 2 h. The reaction
mixture was diluted with H2O and extracted with CH2Cl2
(100 mL). The organic layer was successively washed with
satd. NaHCO3 and water, dried (Na2SO4) and concentrated.
25
m.p. 80°C; [α]D −6.7 (c 1.6, CHCl3); IR (KBr): 3473,
3088, 3063, 3031, 2973, 2933, 2870, 1778, 1744, 1716,
1609, 1508, 1454, 1389, 1232, 1101, 1053, 1027, 971, 965,
1
829, 738, 722, 698 cm−1; H NMR (500 MHz, CDCl3): δ

Glycoconj J (2011) 28:11–19
17
7.37–7.14 (m, 49 H, Ar-H), 6.83 (d, J=9.0 Hz, 2 H, Ar-H),
6.71 (d, J=9.1 Hz, 2 H, Ar-H), 5.76 (d, J=8.5 Hz, 1 H, H-
1A), 5.53 (s, 1 H, PhCH), 5.51 (s, 1 H, PhCH), 5.40–5.39 (m,
1 H, H-2D), 5.34 (br s, 1 H, H-1D), 5.04 (br s, 1 H, H-4B),
4.94 (d, J=2.8 Hz, H-1E), 4.83 (d, J=3.3 Hz, 1 H, H-1C),
4.90–4.87 (m, 2 H, PhCH2), 4.79 (d, J=11.8 Hz, 1 H,
PhCH2), 4.78–4.63 (m, 6 H, PhCH2), 4.69 (d, J=3.6 Hz, 1 H,
H-1B), 4.57–4.52 (m, 5 H, H-2A, H-3A, PhCH2), 4.45-4.36
(m, 1 H, H-3E), 4.35–4.33 (m, 2 H, PhCH2), 4.32 (d, J=
12.9 Hz, 1 H, H-6aA), 4.21 (dd, J=10.0, 3.5 Hz, 1 H, H-3B),
4.16 (m, 1 H, H-5B), 4.12 (m, 1 H, H-5E), 4.07–4.06 (m, 1 H,
H-4D), 3.97–3.78 (m, 8 H, H-2E, H-3C, H-3D, H-4C, H-4E, H-
6bA, H-6abD), 3.75–3.67 (m, 5 H, H-4A, H-5A, H-5C, H-6abC),
3.69 (s, 3 H, OCH3), 3.55 (dd, J=10.2, 3.3 Hz, 1 H, H-2B),
3.52 (m, 1 H, H-5D), 3.32 (dd, J=9.8, 3.6 Hz, 1 H, H-2C),
2.00 (s, 3 H, COCH3), 1.70 (s, 3 H, COCH3), 0.97 (d, J=
6.5 Hz, 3 H, CCH3), 0.63 (d, J=6.5 Hz, 3 H, CCH3); 13C
NMR (125 MHz, CDCl3): δ 170.8, 170.0 (2 COCH3), 167.7
(2 C, Phth), 156.0–114.9 (Ar-C), 102.2 (PhCH), 101.9
poured into cold water and extracted with EtOAc (100 mL).
The organic layer was washed with satd. aq. NaHCO3, dried
(Na2SO4) and concentrated under reduced pressure. The
crude product was purified over SiO2 using hexane-EtOAc
(3:1) as eluant to give pure compound 12 (1.2 g, 76%) as
25
yellow oil. [α]D +11.2 (c 1.6, CHCl3); IR (neat): 3474,
3089, 3063, 3033, 2975, 2933, 2870, 1778, 1745, 1716,
1609, 1508, 1455, 1371, 1312, 1232, 1132, 1101, 1053,
1
1027, 971, 965, 829, 738, 722 cm−1; H NMR (500 MHz,
CDCl3): δ 7.32–7.14 (m, 49 H, Ar-H), 6.83 (d, J=9.0 Hz,
2 H, Ar-H), 6.71 (d, J=9.1 Hz, 2 H, Ar-H), 5.75 (d, J=
8.5 Hz, 1 H, H-1A), 5.52 (s, 1 H, PhCH), 5.50 (s, 1 H,
PhCH), 5.44 (br s, 1 H, H-1D), 5.31 (d, J=3.2 Hz, 1 H, H-
1E), 5.08 (br s, 1 H, H-4B), 4.94 (d, J=11.5 Hz, 1 H,
PhCH2),), 4.87 (d, J=11.3 Hz, 1 H, PhCH2), 4.82 (d, J=
11.3 Hz, 1 H, PhCH2), 4.81–4.79 (m, 2 H, H-1C, H-2D),
4.69–4.64 (m, 6 H, H-1B, PhCH2), 4.62–4.55 (m, 5 H, H-2A,
PhCH2), 4.39–4.36 (m, 3 H, H-3A, PhCH2), 4.26 (d, J=
12.9 Hz, 1 H, H-6aA), 4.17 (m, 1 H, H-5B), 4.11–4.08 (m,
3 H, H-3B, H-4D, H-5E), 4.01 (m, 1 H, H-2E), 3.98–3.95 (m,
2 H, H-3E, H-4A), 3.83–3.81 (m, 2 H, H-3C, H-3D), 3.79-
3.68 (m, 9 H, H-4C, H-4E, H-5A, H-5C, H-6bA, H-6abC, H-
(PhCH), 99.9 ((JC-1/H-1=171 Hz; C-1B), 99.8 (JC-1/H-1
174 Hz; C-1D), 98.7 (2 C, C-1A, C-1C), 94.0 (JC-1/H-1
=
=
172 Hz; C-1E), 82.0 (C-5A), 81.4 (C-5C), 80.2 (C-2E), 79.8
(C-2C), 78.5 (C-5D), 77.2 (C-3D), 76.4 (3 C, C-2B, C-2C, C-
4C), 76.3 (C-3E), 75.3 (PhCH2), 75.2 (PhCH2), 75.0 (C-3A),
74.7 (C-4B), 74.6 (PhCH2), 74.3 (C-3C), 73.9 (PhCH2), 73.5
(PhCH2), 73.4 (PhCH2), 73.0 (PhCH2), 72.9 (PhCH2), 70.8
(C-2D), 69.3 (C-6A), 69.1 (2 C, C-6D, C-6C), 68.8 (C-4D),
67.0 (C-5E), 66.6 (C-4A), 66.3 (C-5B), 65.3 (C-4E), 56.1 (C-
2A), 55.9 (OCH3), 21.2, 20.9 (2 COCH3), 16.9, 16.2 (2
CCH3); MALDI-MS: 1944.7 [M+Na]+; Anal. Calcd. for
6abD), 3.71 (s, 3 H, OCH3), 3.55 (br s, 1 H, H-5D), 3.51 (dd,
J=10.0, 3.3 Hz, 1 H, H-2B), 3.3 (m, 1 H, H-2C), 2.01 (s, 3 H,
COCH3), 1.07 (d, J=6.5 Hz, 3 H, CCH3), 0.58 (d, J=6.5 Hz,
3 H, CCH3); 13C NMR (125 MHz, CDCl3): δ 170.6
(COCH3), 167.9 (2 C, Phth), 156.0–114.9 (Ar-C), 102.3
(PhCH), 102.2 (PhCH), 99.9 (C-1B), 98.7 (C-1A), 98.4 (C-
1C), 97.8 (C-1D), 95.3 (C-1E), 82.1 (C-5A), 81.9 (C-5C), 80.3
(C-2E), 79.3 (2 C, C-2C, C-5D), 78.3 (C-3D), 76.1 (2 C, C-2B,
C-3B), 76.0 (C-4C), 75.6 (PhCH2), 75.3 (PhCH2), 74.9 (C-
3E), 74.6 (C-3A), 74.3 (C-4B), 74.2 (C-2D), 73.8 (2 C, 2
PhCH2), 73.0 (PhCH2), 71.1 (C-3C), 69.5 (C-C6A), 69.4 (C-
6C), 69.1 (2 C, C-4D, C-6D), 67.9 (C-5E), 67.0 (C-4A), 66.3
(C-5B), 64.6 (C-4E), 56.1 (C-2A), 55.9 (OCH3), 21.2,
(COCH3), 16.9, 16.1 (2 CCH3); MALDI-MS: 1927.7
[M+Na]+; Anal. Calcd. for C110H112N4O26 (1904.75): C,
69.31; H, 5.92%; found: C, 69.07; H, 6.18%.
C112H115NO28 (1921.76): C, 69.95; H, 6.03%; found: C,
69.72; H, 6.28%.
4-Methoxyphenyl (2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-
(1→3)-(2-azido-4,6-O-benzylidene-2-deoxy-α-D-glucopyr-
anosyl)-(1→4)-(2,3,6-tri-O-benzyl-α-D-glucopyranosyl)-
(1→3)-(4-O-acetyl-2-O-benzyl-α-L-fucopyranosyl)-(1→3)-
4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyr-
anoside (12) A solution of compound 11 (1.6 g, 0.83
mmol) in 0.05 M CH3ONa in CH3OH (50 mL) was allowed
to stir at room temperature for 2 h and neutralized with
Dowex-50W X8 (H+) resin. The reaction mixture was
filtered and concentrated under reduced pressure. To a
solution of the crude product in anhydrous CH2Cl2 (20 mL)
was added pyridine (5 mL) and the reaction mixture was
cooled to 0°C. Trifluoromethanesulfonic anhydride (triflic
anhydride; 300 μL, 1.78 mmol) was added to the cold
reaction mixture and it was allowed to stir at 0°C for 8 h.
The solvents were removed under reduced pressure and the
crude product was used directly for the next step. To a
soultion of the crude product in dry DMF (5 mL) was
added NaN3 (1.3 g, 20 mmol) and the reaction mixture was
allowed to stir at 70°C for 3 h. The reaction mixture was
4-Methoxyphenyl (α-L-fucopyranosyl)-(1→3)-(2-acet-
amido-2-deoxy-α-D-glucopyranosyl)-(1→4)-(sodium α-D-
glucopyranosyl uronate)-(1→3)-(α-L-fucopyranosyl)-
(1→3)-2-acetamido-2-deoxy-β-D-glucopyranoside (1) To
a solution of compound 12 (1.0 g, 0.52 mmol) in n-butanol
(20 mL) was added ethylene diamine (0.2 mL, 3.0 mmol)
and the reaction mixture was allowed to stir at 90°C for 6
h and the solvents were removed under reduced pressure. A
solution of the crude mass in acetic anhydride-pyridine (3
mL, 1:1 v/v) was kept at room temperature for 2 h and
solvents were removed under reduced pressure and the
crude reaction product was passed through a short pad of
SiO2 using hexane-EtOAc (1:1) as eluant. To a solution of
the acetylated product in CH3OH-EtOAc (10 mL, 1:1 v/v)

18
Glycoconj J (2011) 28:11–19
was added 20% Pd(OH)2-C (150 mg) and the reaction
mixture was allowed to stir at room temperature under a
positive pressure of hydrogen for 6 h. The reaction mixture
was filtered through a Celite® bed and the washed with
CH3OH (100 mL). A solution of the hydrogenolyzed
product in acetic anhydride-pyridine (3 mL, 1:1 v/v) was
kept at room temperature for 2 h and solvents were
removed under reduced pressure. A solution of the crude
mass in 0.1 M sodium methoxide (30 mL) was allowed to
stir at room temperature for 5 h and neutralized with
Dowex-50W X8 (H+) resin. The reaction mixture was
filtered and concentrated under reduced pressure. To a
solution of the crude product in CH2Cl2 (30 mL) and H2O
(5 mL) were added aq. solution of NaBr (2 mL; 1 M), aq.
solution of TBAB (2 mL; 1 M), TEMPO (80 mg, 0.6
mmol), satd. aq. solution of NaHCO3 (10 mL) and 4% aq.
NaOCl (15 mL) in succession and the reaction mixture was
allowed to stir at 0–5°C for 3 h. The reaction mixture was
neutralized with the addition of 1 N aq. HCl solution. To
the reaction mixture were added tert-butanol (20 mL), 2-
methyl-but-2-ene (20 mL; 2 M solution in THF), aq.
solution of NaClO2 (2 g in 10 mL) and aq. solution of
NaH2PO4 (2 g in 10 mL) and the reaction mixture was
allowed to stir at room temperature for 3 h. The reaction
mixture was diluted with satd. aq. NaH2PO4 and extracted
with CH2Cl2 (100 mL). The organic layer was washed with
water, dried (Na2SO4) and concentrated to dryness. The
crude product was passed through a short pad of SiO2 using
CH2Cl2-CH3OH (10:1) as eluant. To a solution of the
oxidized product in CH3OH (10 mL) was added 20% Pd
(OH)2-C (150 mg) and the reaction mixture was allowed to
stir at room temperature under a positive pressure of
hydrogen for 24 h. The reaction mixture was filtered
through a Celite® bed and the washed with CH3OH-H2O
(60 mL; 1:1 v/v) and concentrated under reduced pressure.
The crude product was purified through a Sephadex LH-20
column using CH3OH-H2O (2:1) as eluant to give pure
compound 1 (300 mg, 57%) as a sodium salt as white
(C-4C), 76.1 (C-3C), 73.3 (3 C, C-2C, C-3D, C-5A), 72.7 (2
C, C-4B, C-5C), 72.2 (4 C, C-3E, C-4A, C-4E, C-5D), 70.1 (3
C, C-2B, C-2E, C-3B), 67.4 (C-5B), 66.8 (C-5E), 61.3 (C-2D),
61.1 (2 C, C-6A, C-6D), 56.2 (OCH3), 54.9 (C-2A), 23.7
(COCH3), 20.7 (COCH3), 15.7, 15.6 (2 CCH3); ESI-MS:
1021.3 [M+1]+; Anal. Calcd. for C41H61N2NaO26 (1020.34):
C, 48.24; H, 6.02%; found: C, 48.00; H, 6.29%.
Acknowledgements G. G. thanks CSIR, New Delhi for providing a
Senior Research fellowship. This project was funded by the
Department of Science and Technology (DST), New Delhi through
Ramanna Fellowship (SR/S1/RFPC-06/2006) (A.K.M) and Bose
Institute, Kolkata.
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25
powder. [α]D +8.36 (c 1.6, H2O); IR (KBr): 3410, 2928,
2855, 1709, 1646, 1585, 1566, 1508, 1445, 1392, 1221,
1149, 1082, 1030, 969, 926, 756, 700 cm−1; 1H NMR
(500 MHz, D2O): δ 5.18 (d, J=3.7 Hz, 1 H, H-1B), 5.16
(br s, 1 H, H-1D), 5.15 (br s, 1 H, H-1E), 5.05 (d, J=3.4 Hz,
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9 H, H-2D, H-3B, H-3C, H-4A, H-4C, H-6abC, H-6abD), 3.69
(s, 3 H, OCH3), 3.45–3.41 (m, 4 H, H-4B, H-4E, H-5C, H-
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COCH3), 1.07–1.04 (m, 6 H, 2 CCH3), 13C NMR
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