Synthesis of a tetrasaccharide analog corresponding to the repeating unit of the O-polysaccharide of Salmonella enterica O59: Unexpected stereo outcome in glycosylation

Article abstract of DOI:10.1016/j.carres.2012.01.026

Convergent synthesis of a tetrasaccharide analog corresponding to the repeating unit of the O-polysaccharide of Salmonella enterica O59 is presented. A thioglycoside disaccharide donor was prepared by the glycosylation of two thioglycosides by tuning thei

Full text of DOI:10.1016/j.carres.2012.01.026

Carbohydrate Research 352 (2012) 18–22
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a tetrasaccharide analog corresponding to the repeating
unit of the O-polysaccharide of Salmonella enterica O59: unexpected stereo
outcome in glycosylation
Abhijit Sau a, Rajib Panchadhayee a, Debjani Ghosh b, Anup Kumar Misra a,
⇑
a Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII M, Kolkata 700054, India
b Scottish Church College, 1&3 Urquhart Square, Kolkata 700006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Convergent synthesis of a tetrasaccharide analog corresponding to the repeating unit of the O-polysac-
charide of Salmonella enterica O59 is presented. A thioglycoside disaccharide donor was prepared by
the glycosylation of two thioglycosides by tuning their relative reactivity. An unexpected stereochemical
outcome was observed in a glycosylation using an ethyl 2-O-acetyl-3-O-benzyl-4,6-O-bensylidene-thio-
galactoside donor, where the alpha-galactoside was formed in spite of the presence of the 2-O-acetyl par-
ticipating group.
Received 11 November 2011
Received in revised form 27 January 2012
Accepted 31 January 2012
Available online 10 February 2012
Keywords:
Salmonella enterica
Glycosylation
Ó 2012 Elsevier Ltd. All rights reserved.
O-Polysaccharide
Stereoselectivity
Tetrasaccharide
1. Introduction
carbohydrate based therapeutics.8,9 In order to have better under-
standing of the pathogenic role of the O-antigenic polysaccharide
Salmonella is a Gram-negative facultative rod-shaped bacte-
rium in the family of Enterobacteriaceae, commonly known as
‘enteric’ bacteria.1 Salmonella enterica is responsible for the food
borne gastrointestinal infections causing salmonellosis in ani-
mals and humans.2 Salmonella live in the intestinal tracts of
warm and cold blooded animals. In general, Salmonella enterica
infects cattle and poultry, which act as the reservoir of infections
to humans.3 Approximately 1.5 million cases of salmonellosis
(common symptoms are enteric fever and acute gastroenteritis)
and a significant number of deaths caused due to this infection
are noted annually in the developed and developing countries.4
Salmonella species are divided into a large number of serotypes
based on the structure of their cell wall lipopolysaccharides
(O-antigen) and the flagellar structures (H-antigen).5,6 However,
only few of them are responsible for the infections in humans.
Recently, Perepelov et al. reported the structure of the cell wall
O-polysaccharide of Salmonella enterica O59,7 which contains two
of Salmonella enterica O59, several immunochemical experiments
should be carried out requiring a large quantity of the tetrasaccha-
ride. Since isolation of the polysaccharide fragments from the natu-
ral source can not meet the requirement, a concise chemical
synthetic strategy for the synthesis of the tetrasaccharide and its
close analogs is highly desirable. As a part of the ongoing program
on the synthesis of complex oligosaccharides from the bacterial ori-
gin, synthesis of the tetrasaccharide repeating unit (Fig. 2, structure
1) corresponding to the O-antigenic lipopolysaccharide of Salmo-
nella enterica O59 strain has been undertaken. However, during
the course of the synthesis of the target tetrasaccharide 1, an unu-
sual stereochemical outcome of one of the glycoside formations
was observed. Although using a thiogalactoside donor with a 2-O-
participating group still the disaccharide formed was found to be
alpha-linked. Continuing from this unexpected disaccharide deriva-
tive a tetrasaccharide analog 2 (Fig. 2) related to the O-antigenic
lipopolysaccharide of Salmonella enterica O59 strain was
synthesized.
D-glucosamine, one
D-galactose and one L-rhamnose moieties
(Fig. 1).
Several reports in the recent past elaborately demonstrated the
useful role of O-antigenic polysaccharides in the development of
4)-α-L-Rhap-(1 3)-β-D-GlcpNAc-(1 2)-β-D-Galp-(1 3)-α-D-GlcpNAc-(1
⇑
Figure 1. Structure of the repeating unit of the O-polysaccharide of Salmonella
enterica O59.
Corresponding author. Tel.: +91 33 2569 3240; fax: +91 33 2355 3886.
E-mail address: akmisra69@gmail.com (A.K. Misra).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2012.01.026

A. Sau et al. / Carbohydrate Research 352 (2012) 18–22
19
OH
OH
HO
HO
OPMP
AcHN
O
O
OH
B
OH
B
HO
HO
O
HO
OPMP
A
AcHN
O
O
HO
O
HO
O
O
C
O
HO
C
O
A
O
OH
O
NHAc
NHAc
O
OH
O
D
D
HO
HO
HO
HO
1
2
PMP: 4-methoxyphenyl
OH
OH
Figure 2. Chemical structure of the alpha –p-methoxybenzyl glycoside of the tetrasaccharide repeating unit of the O-polysaccharide of Salmonella enterica O59 (1) and
structure of the synthesized tetrasaccharide 2 related to tetrasaccharide 1.
Ph
O
SEt
Ph
O
O
Ph
SEt
O
O
O
O
O
A
O
O
D
HO
C
B
BnO
SEt
HO
AcO
N
3OPMP
AcO
NPhth
AcO
OAc
4
6
7
5
Figure 3. Monosaccharide intermediates used for the synthesis of compound 2.
glycosylations. It is presumed that a five-member twisted boat
transition state may form during the activation of the thioglycoside
with NIS–TfOH, which forces the nucleophile to approach from the
1,2-cis orientation and thus furnishes 1,2-cis glycosylated product.
In earlier reports by Satoh et al.,17 Manabe et al.,18 Oscarson et al.19
and Crich et al.20 it was considered that endo-cyclic post glycosyl-
ation anomerization plays the pivotal role in the formation of 1,2-
cis glycosylation products. Recently, Oscarson and co-workers21 re-
ported the anomerization of disaccharides by kinetic measure-
ments. However, in this particular case such possibilities may be
excluded since the functional groups present in the glycosyl donor
are different. Although the formation of compound 8 was unex-
pected and can not be used for the synthesis of the target tetrasac-
charide 1, we planned to use compound 8 in the preparation of an
analogous tetrasaccharide 2 related to the target compound 1.
Analogous compounds are very important in evaluating their bio-
logical potential in comparison to the original compounds. Thus
compound 8 was deacetylated using sodium methoxide to give
disaccharide derivative 9 in quantitative yield.
2. Results and discussion
In the initial attempt to synthesize compound 1, a number of
suitably functionalized monosaccharide intermediates 3,10 4, 5,11
6
12, and 712 (Fig. 3) were prepared from the commercially available
reducing sugars using earlier reported reaction conditions. A con-
vergent strategy involving [2+2] glycosylation has been adopted
for the synthesis of compound 1. Compound 4 was prepared in
77% yield from compound 3 using a two step sequence involving
de-acetylation and iodine catalyzed 4,6-O-benzylidene acetal for-
mation13 (Scheme 1). Iodonium ion promoted stereoselective gly-
cosylation14 of compound
4 with thioglycoside 5 using N-
iodosuccinimide (NIS) and trifluoromethane sulfonic acid (TfOH)
furnished compound 8 in 77% yield having a newly formed 1,2-
cis glycosyl linkage. Formation of 1,2-cis glycoside was confirmed
from its spectral analysis [signals at d 5.68 (d, J = 4.0 Hz, H-1B),
5.52 (d, J = 3.5 Hz, H-1A) in the 1H NMR and d 98.6 (C-1A), 97.4
(C-1B) in the 13C NMR spectra]. Formation of compound 8 with
1,2-cis glycosyl linkage using 2-O-acetylated D-galactosyl thioethyl
In another experiment, stereoselective glycosylation of thiogly-
coside 6 and thioglycoside 7 in the presence of a combination of
NIS–TfOH14 furnished disaccharide derivative 10 in an 81% yield.
Appearance of signature peaks in the NMR spectra [signals at d
5.37 (d, J = 10.5 Hz, H-1C), 4.51 (br s, H-1D) in the 1H NMR and d
97.3 (C-1D), 81.9 (C-1C) in the 13C NMR spectra] confirmed the for-
mation of compound 10. Although both compounds 6 and 7 are
thioethyl glycosides and can be activated by the combination of
NIS–TfOH, the selective activation of compound 7 was achieved be-
cause of the fact that deoxy sugars are more highly reactive than
the amino sugars, which has been established in several earlier re-
ports by Wong and co-workers using relative reactivity values.22,23
Presence of a deactivating N-phthalimido group at C-2 position of
compound 6 induces disarming effect to act as glycosyl acceptor
glycoside donor was unexpected due to the well known concept
that the presence of an acyl protection group on 2-OH of a glycosyl
donor commonly induces the exclusive formation of a 1,2-trans
glycoside because of the neighboring group participation ef-
fect.15,16 Using other 2-O-acetylated glycosyl donors (e.g., thio-
phenyl glycoside, trichloroacetimidate derivative) showed similar
unexpected stereo outcome of the glycosylation. In a separate
experiment, reaction of the same glycosyl acceptor with ethyl
2,3-di-O-acetyl-4,6-O-benzylidene-1-thio-b-D-galactopyranoside,
exclusively furnished 1,2-trans glycosylated product (80%). In both
galactosyl thioglycoside donors 4,6-O-benzylidene acetal is pres-
ent, but the difference in 3-O-substituents (benzyl and acetyl) di-
rects the stereochemical outcome of the glycosylation product.
This observation indicated that the presence of 3-O-benzyl ether
in the thioglycoside donor controls the stereo-outcome of the
in the presence of a comparatively activated 6-deoxy-L-sugar de-
rived thioglycoside 7. This kind of selective activation has also been
documented by Demchenko and co-workers24 and Bols and co-
workers25 considering super armed/disarmed glycosylation ap-
proach. Iodonium ion promoted stereoselective glycosylation of
disaccharide 9 with disaccharide 10 using NIS–TfOH combination
furnished tetrasaccharide derivative 11 in a 74% yield. Formation
of compound 11 was supported by it’s spectral analysis [signals
at d 5.55 (d, J = 4.0 Hz, H-1B), 5.48 (d, J = 3.5 Hz, H-1A), 5.39 (d,
J = 8.0 Hz, H-1C), 4.45 (d, J = 1.5 Hz, H-1D) in the 1H NMR and d
OAc
Ph
O
O
O
O
AcO
AcO
a, b
HO
N
N
3OPMP
3OPMP
4
3
Scheme 1. Reagents and conditions: (a) 0.1 M CH3ONa, CH3OH, room temperature,
2 h; (b) benzaldehyde dimethyl acetal, I2, CH3CN, 50 °C, 8 h, 77%.

20
A. Sau et al. / Carbohydrate Research 352 (2012) 18–22
99.6 (C-1B), 98.6 (C-1A), 98.0 (C-1C), 97.1 (C-1D) in the 13C NMR
spectra]. Compound 11 was subjected to a series of reactions
involving (a) removal of N-phthalimido group followed by N-acet-
ylation,26 (b) hydrogenolysis27 followed by N-acetylation and (c)
saponification to furnish compound 2 in a 56% over all yield. Pres-
ence of signals in the NMR spectra confirmed its formation [signals
at d 5.62 (d, J = 4.0 Hz, H-1B), 5.42 (d, J = 8.5 Hz, H-1C), 5.31 (d,
J = 3.5 Hz, H-1A), 4.56 (d, J = 1.5 Hz, H-1D) in the 1H NMR and d
102.9 (C-1D), 101.7 (C-1C), 100.3 (C-1B), 99.1 (C-1A) in the 13C
NMR spectra] (Scheme 2). A comparison of the chemical shifts of
the anomeric centers of the synthesized tetrasaccharide as its 4-
methoxyphenyl glycoside (2) with the tetrasaccharide fragment
corresponding to the O-antigenic lipopolysaccharide of Salmonella
enterica O59 is presented in Table 1. A slight deviation of the chem-
ical shifts in the synthesized compound 2 may be due to the pres-
ence of a 4-methoxyphenyl group at the reducing end whereas the
isolated tetrasaccharide was a hemiacetal. From the chemical shift
Table 1
Comparison of the NMR chemical shifts (d, ppm) and coupling constants (J in Hz) of
the anomeric centers of compound 2 and the tetrasaccharide repeating unit isolated
from the natural source7
Sugar residue
H-1 (d, ppm)
Compd 2
C-1 (d, ppm)
Isolated7 Compd 2 Isolated7
?3)-
(A)
?2)-
a
-
D
-GlcpNAc
5.31 (J = 3.5 Hz)
4.99
99.1
99.2
a
/b- -Galp (B)
D
5.62
4.56 (b)
100.3
102.2
(J = 4.0 Hz)(
-GlcpNAc (C) 5.42 (J = 8.5 Hz)
a-L-Rhap (D) 4.56 (J = 1.5 Hz)
a)
?3)-b-
D
4.80
4.87
101.7
102.9
102.5
102.2
on a hot plate. Silica gel 230–400 mesh was used for column chro-
matography. 1H and 13C NMR, DEPT 135, 2D COSY, and HSQC NMR
spectra were recorded on Brucker Avance DRX 500 MHz spectrom-
eters using CDCl3 and CD3OD as solvents and TMS as internal ref-
erence unless stated otherwise. Chemical shift values are
expressed in d ppm. ESI-MS were recorded on a Micromass mass
spectrometer. Elementary analysis was carried out on Carlo Erba
analyzer. Optical rotations were measured at 25 °C on a Jasco P-
2000 polarimeter. Commercially available grades of organic sol-
vents of adequate purity are used in all reactions.
values it is clear that D-galactose moiety is 1,2-cis linked in com-
pound 2.
3. Conclusion
In conclusion, it was observed that the stereo chemical outcome
of the glycosylation is strongly dependent on the functionalities
present in the donor and acceptor and often deviates from the gen-
erally accepted explanation of neighboring group participating ef-
fect for the 1,2-trans glycoside formation. A tetrasaccharide analog
related to the repeating unit of the O-polysaccharide of Salmonella
enterica O59 has been synthesized using a [2+2] block glycosyla-
tion strategy. An intermediate disaccharide donor was prepared
by glycosylation of two thioglycosides by tuning their relative
reactivity. All intermediates were obtained in a high yield.
4.2. 4-Methoxyphenyl 2-azido-4,6-O-benzylidene-2-deoxy-a-D-
glucopyranoside (4)
A solution of compound 3 (4.0 g, 9.15 mmol) in 0.1 M CH3ONa
in CH3OH (30 mL) was allowed to stir at room temperature for
2 h, neutralized with Dowex 50W X8 (H+) resin and the solvents
were removed under reduced pressure. To a solution of the product
in anhydrous CH3CN (25 mL) were added benzaldehyde dimethyl-
acetal (2.7 mL, 18.0 mmol) and iodine (500 mg, 1.97 mmol) and the
reaction mixture was allowed to stir at 50 °C for 8 h. To the reac-
tion mixture was added Et3N (2 mL) and it was evaporated to dry-
ness under reduced pressure. The crude mass was dissolved in
CH2Cl2 (150 mL) and successively washed with 5% Na2S2O3, satd
NaHCO3, and water. The organic layer was dried (Na2SO4) and con-
centrated under reduced pressure to give the crude product, which
was purified over SiO2 using hexane–EtOAc (4:1) as eluant to give
4. Experimental
4.1. 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 sulfate (2% Ce(SO4)2 in 2 N H2SO4)-sprayed plates
Ph
O
O
Ph
D
O
O
O
a
a
C
O
6 + 7
SEt
NPhth
O
4 + 5
X
B
BnO
OPMP
N3
O
RO
O
A
AcO
O
O
AcO
O
OAc
10
Ph
8:
R = Ac
R = H
a
b
9:
Ph
Ph
O
O
OPMP
N3
O
O
O
B
BnO
O
A
O
O
O
AcO
Ph
O
B
BnO
OPMP
O
c, d, e
Ph
N3
O
O
2
O
O
C
A
Expected
O
O
O
NPhth
O
Ph
O
D
AcO
AcO
11
OAc
Scheme 2. Reagents and conditions: (a) N-iodosuccinimide, TfOH, CH2Cl2, MS 4 Å, ꢀ30 °C, 45 min, 77% for compound 8, 81% for compound 10, and 74% for compound 11; (b)
0.1 M CH3ONa, CH3OH, room temperature, 2 h, quantitative; (c) (i) NH2NH2ꢁH2O, EtOH, 80 °C, 8 h; (ii) acetic anhydride, pyridine, room temperature, 1 h; (d) (i) H2, 20%
Pd(OH)2-C, CH3OH, 24 h, room temperature; (ii) acetic anhydride, pyridine, room temperature, 1 h; (e) 0.1 M CH3ONa, CH3OH, room temperature, 2 h, 56% over all.

A. Sau et al. / Carbohydrate Research 352 (2012) 18–22
21
pure compound 4 (2.8 g, 77%). Yellow oil; ½a D25
ꢂ
+120 (c 1.2, CHCl3);
IR (neat): 2963, 2944, 2116, 1752, 151, 1230, 1044, 1035, 819, 786,
4A), 76.4 (C-4B), 73.6 (C-3B), 73.0 (C-3A), 71.4 (PhCH2), 69.8 (C-
6A), 68.6 (C-6B), 67.9 (C-2B), 63.7 (C-5B), 63.1 (C-5A), 61.9 (C-2A),
55.7 (OCH3); ESI-MS: 762.2 [M+Na]+; Anal. Calcd for C40H41N3O11
(739.27): C, 64.94; H, 5.59. Found: C, 64.72; H, 5.85.
543 cmꢀ1 1H NMR (CDCl3, 500 MHz): d 7.49–7.36 (m, 5H, Ar-H),
;
7.01 (d, J = 9.0 Hz, 2H, Ar-H), 6.85 (d, J = 9.0 Hz, 2H, Ar-H), 5.53 (s,
1H, PhCH), 5.39 (d, J = 3.3 Hz, 1H, H-1), 4.38–4.32 (m, 1H, H-6a),
4.25–4.20 (m, 1H, H-6b), 4.06–3.97 (m, 1H, H-5), 3.76 (s, 3H,
OCH3), 3.71 (t, J = 10.2 Hz each, 1H, H-4), 3.55 (t, J = 9.6 Hz each,
1H, H-3), 3.36 (dd, J = 9.9, 3.6 Hz, 1H, H-2); 13C NMR (CDCl3,
125 MHz): d 155.6–114.7 (Ar-C), 102.1 (PhCH), 98.3 (C-1), 81.7,
68.8, 68.7, 66.3, 63.1, 55.7; ESI-MS: 422.1 [M+Na]+; Anal. Calcd
for C20H21N3O6 (399.14): C, 60.14; H, 5.30. Found: C, 60.30; H, 5.50.
4.5. Ethyl (2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-(1?3)-4,6-
O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-b-D-
glucopyranoside (10)
To a solution of compound 6 (1.0 g, 2.26 mmol) and compound
7 (840 mg, 2.51 mmol) in anhydrous CH2Cl2 (5 mL) was added MS
4 Å (1.0 g) and the reaction mixture was stirred under argon at
room temperature for 30 min. The reaction mixture was cooled
4.3. 4-Methoxyphenyl (2-O-acetyl-3-O-benzyl-4,6-O-
benzylidene-
benzylidene-2-deoxy-
a
-
D
-galactopyranosyl)-(1?3)-2-azido-4,6-O-
-glucopyranoside (8)
to ꢀ30 °C and NIS (550.0 mg, 2.44 mmol) and TfOH (10
lL) were
a-
D
added to it. After stirring at same temperature for 45 min the reac-
tion mixture was filtered through a CeliteÒ bed and washed with
CH2Cl2 (50 mL). The organic layer was successively washed with
5% Na2S2O3, satd. NaHCO3 and water, dried (Na2SO4), and concen-
trated under reduced pressure. The crude product was purified
over SiO2 using hexane–EtOAc (5:1) as eluant to give pure com-
To a solution of compound 4 (1.0 g, 2.5 mmol) and compound 5
(1.3 g, 2.9 mmol) in anhydrous CH2Cl2 (10 mL) was added MS 4 Å
(1.0 g) and the reaction mixture was stirred under argon at room
temperature for 30 min. The reaction mixture was cooled to
ꢀ30 °C and N-iodosuccinimide (NIS; 780 mg, 3.46 mmol) and tri-
pound 10 (1.3 g, 81%). Yellow oil; ½a D25
ꢀ26 (c 1.2, CHCl3); IR (neat):
ꢂ
fluoromethane sulfonic acid (TfOH; 15
lL) were added to it. After
2983, 1751, 1716, 1383, 1224, 1096, 1045, 721, 545 cmꢀ1; 1H NMR
(CDCl3, 500 MHz): d 7.73–7.31 (m, 9H, Ar-H), 5.57 (s, 1H, PhCH),
5.37 (d, J = 10.5 Hz, 1H, H-1C), 5.20 (dd, J = 10.0, 3.0 Hz, 1H, H-3D),
4.80 (t, J = 10.0 Hz each 1H, H-4D), 4.71–4.70 (m, 1H, H-2D), 4.66
(t, J = 9.5 Hz each, 1H, H-3C), 4.51 (br s, 1H, H-1D), 4.42–4.39 (m,
1H, H-6ac), 4.38 (t, J = 10.5 Hz each,1H, H-2C), 4.10–3.97 (m, 1H,
H-5D), 3.83–3.79 (m, 1H, H-6bc), 3.77–3.71 (m, 2H, H-4C, H-5C),
2.68–2.60 (m, 2H, SCH2CH3), 1.93, 1.90, 1.76 (3 s, 9H, 3 COCH3),
1.71 (t, J = 7.5 Hz each, 3H, SCH2CH3), 0.57 (d, J = 6.5 Hz, 3H,
CCH3); 13C NMR (CDCl3, 125 MHz): d 169.8, 169.6, 169.9, 169.3
(COCH3), 137.0–123.2 (Ar-C), 102.0 (PhCH), 97.3 (C-1D), 81.9 (C-
1C), 80.2 (C-5C), 75.3 (C-3C), 71.1 (C-4D), 70.8 (C-4C), 70.2 (C-2D),
68.6 (C-6C), 68.2 (C-3D), 66.4 (C-5D), 55.1 (C-2C), 24.1 (SCH2CH3),
20.7, 20.6, 20.4 (3 COCH3), 16.3 (CCH3), 14.8 (SCH2CH3); ESI-MS:
736.2 [M+Na]+; Anal. Calcd for C35H39NO13S (713.21): C, 58.90; H,
5.51. Found: C, 58.73; H, 5.72.
stirring at the same temperature for 45 min the reaction mixture
was filtered through a CeliteÒ bed and washed with CH2Cl2
(100 mL). The organic layer was successively washed with 5%
Na2S2O3, satd. NaHCO3, and water, dried (Na2SO4) and concen-
trated under reduced pressure. The crude product was purified
over SiO2 using hexane–EtOAc (6:1) as eluant to give pure com-
pound 8 (1.5 g, 77%). Yellow oil; ½a D25
ꢂ
+109 (c 1.2, CHCl3); IR (neat):
2920, 2864, 2106, 1743, 1508, 1235, 1093, 1029, 744, 698 cm-1; 1H
NMR (CDCl3, 500 MHz): d 7.33–7.26 (m, 15H, Ar-H), 7.04 (d, 9.0 Hz,
2H, Ar-H), 6.86 (d, J = 9.0 Hz, 2H, Ar-H), 5.68 (d, J = 4.0 Hz, 1H, H-
1B), 5.52 (d, J = 3.5 Hz, 1H, H-1A), 5.50 (s, 1H, PhCH), 5.47 (s, 1H,
PhCH), 5.44 (dd, J = 10.5, 3.5 Hz, 1H, H-2B), 4.73–4.67 (ABq,
J = 12.0 Hz each, 2H, PhCH2), 4.54 (t, J = 9.5 Hz each, 1H, H-3A),
4.34–4.31 (m, 1H, H-6aA), 4.26 (d, J = 3.5 Hz, 1H, H-4B), 4.25–4.22
(m, 1H, H-6bA), 4.12–4.07 (m, 1H, H-5A), 4.05 (dd, J = 10.5, 3.0 Hz,
1H, H-3B), 4.03–4.02 (m, 1H, H-5B), 4.00–3.98 (m, 1H, H-6aB),
3.78 (s, 3H, OCH3), 3.77–3.73 (m, 2H, H-4A, H-6bB), 3.29 (dd,
J = 10.5, 3.5 Hz, 1H, H-2A), 1.74 (s, 3H, COCH3); 13C NMR (CDCl3,
125 MHz): d 169.4 (COCH3), 155.6–114.8 (Ar-C), 101.9 (PhCH),
101.0 (PhCH), 98.6 (C-1A), 97.4 (C-1B), 82.1 (C-4A), 74.2 (C-4B),
73.4 (C-3B), 72.6 (C-3A), 71.7 (PhCH2), 69.6 (C-6A), 69.1 (C-2B),
68.7 (C-6B), 63.4 (C-5B), 63.0 (C-5A), 61.4 (C-2A), 55.6 (OCH3), 20.9
(COCH3); ESI-MS: 804.2 [M+Na]+; Anal. Calcd for C42H43N3O12
(781.28): C, 64.52; H, 5.54. Found: C, 64.30; H, 5.75.
4.6. 4-Methoxyphenyl (2,3,4-tri-O-acetyl-
a-L-
rhamnopyranosyl)-(1?3)-(4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-
D
-glucopyranosyl)-(1?2)-(3-O-benzyl-4,6-O-
-galactopyranosyl)-(1?3)-2-azido-4,6-O-
benzylidene-
a-
D
benzylidene-2-deoxy-a-D-glucopyranoside (11)
To a solution of compound 9 (1.0 g, 1.35 mmol) and compound
10 (1.0 g, 1.40 mmol) in anhydrous CH2Cl2 (5 mL) was added MS
4 Å (1.0 g) and the reaction mixture was stirred under argon at
room temperature for 30 min. The reaction mixture was cooled
4.4. 4-Methoxyphenyl (3-O-benzyl-4,6-O-benzylidene-
galactopyranosyl)-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-
-glucopyranoside (9)
a-D-
to ꢀ30 °C and NIS (350.0 mg, 1.55 mmol) and TfOH (5
lL) were
a-
D
added to it. After stirring at the same temperature for 45 min the
reaction mixture was filtered through a CeliteÒ bed and washed
with CH2Cl2 (50 mL). The organic layer was successively washed
with 5% Na2S2O3, satd NaHCO3 and water, dried (Na2SO4), and con-
centrated under reduced pressure. The crude product was purified
over SiO2 using hexane–EtOAc (4:1) as eluant to give pure com-
A solution of compound 8 (1.3 g, 1.66 mmol) in 0.1 M CH3ONa
in CH3OH (15 mL) was allowed to be stirred at room temperature
and neutralized with Dowex 50W X8 (H+) resin. The reaction mix-
ture was filtered, concentrated, and passed through a short pad of
SiO2 to give pure compound 9 (1.2 g, 98%). Yellow oil; ½a D25
ꢂ
+89 (c
pound 11 (1.4 g, 74%). Yellow oil; ½a D25
ꢂ
+86 (c 1.2, CHCl3); IR (neat):
1.2, CHCl3); IR (neat): 3529, 2924, 2104, 1508, 1370, 1210, 1027,
3475, 2932, 2107, 1751, 1717, 1508, 1387, 1223, 1045, 698 cmꢀ1
;
743, 697 cm-1
;
1H NMR (CDCl3, 500 MHz): 7.52–7.25 (m, 15H, Ar-
1H NMR (CDCl3, 500 MHz): d 7.71–7.18 (m, 24H, Ar-H), 6.12 (s,
1H, PhCH), 5.55 (d, J = 4.0 Hz, 1H, H-1B), 5.48 (d, J = 3.5 Hz, 1H, H-
1A), 5.39 (d, J = 8.0 Hz, 1H, H-1C), 5.30 (dd, J = 10.0, 3.5 Hz, 1H, H-
3D), 5.24 (s, 1H, PhCH), 5.22 (s, 1H, PhCH), 4.84 (t, J = 10.0 Hz each,
1H, H-4D), 4.75–4.74 (m, 1H, H-2D), 4.53–4.46 (m, 2H, H-3A, H-4C),
4.45 (d, J = 1.5 Hz, 1H, H-1D), 4.41 (d, J = 12.8 Hz, 1H, PhCH2), 4.30–
4.27 (m, 2H, H-2B, H-6aB), 4.25–4.22 (m, 1H, H-6aA), 4.11–4.09 (m,
1H, H-6bA), 4.06–4.00 (m, 3H, H-3C, H-6aC, PhCH2), 3.98–3.91 (m,
4H, H-2C, H-5C, H-5D, H-6bB), 3.88–3.87 (d, J = 3.5 Hz, 1H, H-4B),
H), 7.04 (d, J = 7.5 Hz, 2H, Ar-H), 6.85 (d, J = 7.5 Hz, 2H, Ar-H),
5.62 (br s, 1H, H-1B), 5.59 (s, 1H, PhCH), 5.50 (br s, 1H, H-1A),
5.45 (s, 1H, PhCH), 4.73 (br s, 2H, PhCH2), 4.54 (t, J = 8.0 Hz, 1H,
H-3A), 4.35–4.34 (m, 1H, H-6aA), 4.27–4.26 (m, 3H, H-3B, H-4B, H-
6bA), 4.07–4.03 (m, 2H, H-3B, H-5A), 3.39 (br s, 1H, H-5B), 3.88–
3.72 (m, 3H, H-4A, H-6abB), 3.74 (s, 3H, OCH3), 3.33 (dd, J = 9.5,
3.0 Hz, 1H, H-2A); 13C NMR (CDCl3, 125 MHz): d 155.7–114.8 (Ar-
C), 101.6 (PhCH), 100.8 (PhCH), 100.3 (C-1A), 98.5 (C-1B), 82.2 (C-

22
A. Sau et al. / Carbohydrate Research 352 (2012) 18–22
3.81 (br s, 1H, H-5B), 3.78 (s, 3H, OCH3), 3.65 (dd, J = 10.5, Hz, 1H,
H-3B), 3.43–3.37 (m, 1H, H-5A), 3.20 (dd, J = 10.0, 3.5 Hz, 1H, H-
2A), 3.05 (t, J = 10.0 Hz each, 1H, H-6bC), 2.53 (t, J = 10.0 Hz each,
1H, H-4A), 1.99, 1.95, 1.79 (3 s, 9H, 3 COCH3), 0.57 (d, J = 6.5 Hz,
1H, CCH3); 13C NMR (CDCl3, 125 MHz): d 169.9, 169.8, 169.4 (3
COCH3), 167.9, 167.8 (PhthCO), 155.6–114.7 (Ar-C), 101.4 (PhCH),
101.2 (PhCH), 101.0 (PhCH), 99.6 (C-1B), 98.6 (C-1A), 98.0 (C-1C),
97.1 (C-1D), 82.0 (C-5C), 79.3 (C-4A), 75.9 (C-3B), 74.3 (C-4B), 74.0
(C-4C), 72.4 (C-3A), 71.8 (PhCH2), 71.1 (C-2B), 71.0 (C-4D), 70.3 (C-
2D), 69.7 (C-6B), 68.7 (C-6A), 68.5 (C-3D), 68.0 (C-6c), 66.3 (C-5D),
65.4 (C-5A), 63.2 (C-3C), 63.0 (C-5B), 61.7 (C-2A), 57.0 (C-2C), 55.6
(OCH3), 20.7, 20.6, 20.5 (3 COCH3), 16.4 (CCH3); ESI-MS: 1413.4
[M+Na]+; Anal. Calcd for C73H74N4O24 (1390.47): C, 63.02; H,
5.36. Found: C, 63.21; H, 5.60.
2 COCH3), 17.8 (CCH3); ESI-MS: 861.3 [M+Na]+; Anal. Calcd for
35H54N2O21 (838.32): C, 50.12; H, 6.49. Found: C, 49.94; H, 6.72.
C
Acknowledgements
A.S. and R.P. thank the CSIR, New Delhi for providing Senior Re-
search Fellowships. This project was funded by the Department of
Science and Technology (DST), New Delhi (SR/S1/OC-83/2010) and
the Bose Institute, Kolkata.
Supplementary data
Supplementary data associated (copies of the 1D and 2D NMR
spectra of compounds 8–11 and 2) with this article can be found,
in the online version, at doi:10.1016/j.carres.2012.01.026.
4.7. 4-Methoxyphenyl (
a-L-rhamnopyranosyl)-(1?3)-(2-
acetamido-2-deoxy-b- -glucopyranosyl)-(1?2)-(a-D-
galactopyranosyl)-(1?3)-2-acetamido-2-deoxy-a-D-
D
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glucopyranoside (2)
To a solution of compound 11 (1.0 g, 0.72 mmol) in EtOH
(20 mL) was added hydrazine monohydrate (0.2 mL, 4.12 mmol)
and the reaction mixture was allowed to stir at 80 °C for 8 h. After
removal of the solvents the crude mass was dissolved in acetic
anhydride-pyridine (5 mL; 1:1 v/v) and kept at room temperature
for 1 h. The solvents were removed under reduced pressure to give
the crude acetylated product. To a solution of the crude mass in
CH3OH (10 mL) was added 20% Pd(OH)2-C (200 mg) and the reac-
tion mixture was stirred at room temperature for 24 h. The reac-
tion mixture was filtered through a CeliteÒ bed and evaporated
to dryness. A solution of the crude product in acetic anhydride-pyr-
idine (3 mL; 1:1 v/v) was kept at room temperature for 1 h and
concentrated under reduced pressure. A solution of the crude prod-
uct in 0.1 M CH3ONa in CH3OH (10 mL) was allowed to stir at room
temperature for 2 h and neutralized with Dowex 50W X8 (H+) re-
sin. The reaction mixture was filtered and concentrated to dryness
to give compound 2, which was passed through a Sephadex LH-20
column using CH3OH–H2O (4:1) as eluant to give pure compound 2
(340 mg, 56%). White powder; ½a D25
ꢂ
+78 (c 1.3, CH3OH); IR (KBr):
3423, 2928, 1767, 1655, 1578, 1211, 967 cmꢀ1
;
1H NMR (CD3OD,
500 MHz): d 7.07 (d, J = 9.0 Hz, 2H, Ar-H), 6.84 (d, J = 9.0 Hz, 2H,
Ar-H), 5.62 (d, J = 4.0 Hz, 1H, H-1B), 5.42 (d, J = 8.5 Hz, 1H, H-1C),
5.31 (d, J = 3.5 Hz, 1H, H-1A), 4.56 (d, J = 1.5 Hz, 1H, H-1D), 4.41
(dd, J = 8.0 Hz each, 1H, H-3C), 4.19 (dd, J = 8.5 Hz each, 1H, H-2C),
4.15 (dd, J = 10.5, 3.5 Hz, 1H, H-2A), 4.13–4.08 (m, 1H, H-5A),
4.03–3.98 (m, 3H, H-2B, H-4B, H-6aA), 3.87–3.81 (m, 1H, H-6bA),
3.80–3.74 (m, 6H, H-3A, H-3B, H-3D, H-5C, H-6abB), 3.73 (s, 3H,
OCH3), 3.72–3.65 (m, 2H, H-4D, H-6ac), 3.58–3.49 (m, 4H, H-4C,
H-5B, H-5D, H-6bc), 3.39–3.37 (m, 1H, H-2D), 3.28 (t, J = 9.5 Hz each,
1H, H-4A), 2.14, 1.94 (2 s, 6H, 2 COCH3), 1.21 (d, J = 6.0 Hz, 3H,
CCH3); 13C NMR (CD3OD, 125 MHz): d 173.7 (2 C, 2 COCH3),
156.8–115.6 (Ar-C), 102.9 (C-1D), 101.7 (C-1C), 100.3 (C-1B), 99.1
(C-1A), 81.1 (C-3C), 79.8 (C-5A), 79.6 (C-2B), 78.3 (C-5B), 76.5 (C-
5D), 74.1 (C-3A), 73.5 (C-4A), 72.5 (C-2D), 72.2 (C-4B), 71.9 (C-4B),
71.8 (C-5C), 71.1 (C-4C), 70.7 (C-4D), 68.3 (C-3D), 62.6 (2 C, C-6A,
C-6B), 62.4 (C-6C), 57.3 (C-2A), 56.0 (OCH3), 53.6 (C-2C), 22.8 (2 C,
27. Pearlman, W. M. Tetrahedron Lett. 1967, 8, 1663–1664.