Synthesis of a pentasaccharide repeating unit of the O-antigen of enteroadherent Escherichia coli O154 strain

Article abstract of DOI:10.1016/j.tetasy.2014.03.007

A straightforward linear synthetic strategy has been developed for the synthesis of the pentasaccharide repeating unit of the cell wall O-antigenic polysaccharide of enteroadherent Escherichia coli O154 strain. Newly developed glycosylation conditions using glycosyl trichloroacetimidate derivatives as glycosyl donors and nitrosyl tetrafluoroborate as the glycosylation activator have been used in all of the glycosylation reactions throughout the synthetic scheme. The stereochemical outcomes of the glycosylations were excellent and the yields were very good.

Full text of DOI:10.1016/j.tetasy.2014.03.007

Tetrahedron: Asymmetry 25 (2014) 632–636
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of a pentasaccharide repeating unit of the O-antigen of
enteroadherent Escherichia coli O154 strain
⇑
Manas Jana , Abhijit Sau , Anup Kumar Misra
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata 700054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A straightforward linear synthetic strategy has been developed for the synthesis of the pentasaccharide
repeating unit of the cell wall O-antigenic polysaccharide of enteroadherent Escherichia coli O154 strain.
Newly developed glycosylation conditions using glycosyl trichloroacetimidate derivatives as glycosyl
donors and nitrosyl tetrafluoroborate as the glycosylation activator have been used in all of the glycosyl-
ation reactions throughout the synthetic scheme. The stereochemical outcomes of the glycosylations
were excellent and the yields were very good.
Received 29 January 2014
Accepted 11 March 2014
Available online 4 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Since the oligosaccharide repeating unit of the polysaccharide
shows similar antigenicity, it would be worthwhile developing a
Enteric infections such as diarrhea, gastroenteritis, colitis, and
hemorrhagic uremic syndrome are serious concerns in developing
countries, which are spread through contaminated food and
water.1–3 Most of the diarrheal outbreaks reported so far are asso-
ciated with pathogenic Escherichia coli (E. coli) strains.4,5 Although
most E. coli strains are found in the human colonic flora as com-
mensal microorganisms, certain strains acquire virulence factors
and cause a number of enteric infections.6 Diarrheal E. coli strains
are classified in several classes based on the nature of their infec-
tions such as, enteroinvasive E. coli (EIEC), enteropathogenic
E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroadhesive
E. coli (EAEC) etc.7 Bacterial cell wall O-polysaccharides are respon-
sible for their pathogenic actions and play important roles in the
initial stage of bacterial infection to the host. The emergence of
multi-drug resistant bacterial strains poses extra challenges in
drug discovery programs toward the development of antibacterial
agents and their efficacy.8 Since cell wall polysaccharides possess
high antigenicity, several attempts have been made to develop
antibacterial vaccine candidates using cell wall O-antigen polysac-
charides.9,10 Recently, Perepelov et al. reported on the structure of
the pentasaccharide repeating unit of the cell wall O-antigen of
enteroadherent E. coli O154 strain, which has been isolated from
diarrheal patients from Brazil, Myanmar, and Japan.11 The repeat-
glycoconjugate derivative based on the pentasaccharide repeating
unit of the O-antigen of E. coli O154. In order to prepare
glycoconjugate derivatives for biological evaluation, a significant
quantity of oligosaccharide with adequate purity is required,
which is practically inconvenient to isolate from natural sources.
Hence, the development of a chemical synthetic strategy would
be useful to gain access to a considerable amount of the required
oligosaccharide with high purity. Therefore, a straightforward
HO
OH
HO
O
A
O
B O
HO
AcHN
C
O
AcHN
OPMP
HO
O
O
HO
HO
HO
HO
O
E
D O
HO
O
HO
PMP: p-methoxyphenyl
1
Ph
CCl3
O
O
N3
O
AcO
AcO
AcO
ing unit consists of three L-rhamnose, one D-galactosamine, and
one mannosamine units, which are alpha glycosidically linked.
O
NH
OBn
O
O
BnO
NH
CCl3
HO
AcO
N3
OPMP
O
3
2
4
⇑
Corresponding author. Tel.: +91 33 2569 3240; fax: +91 33 2355 3886.
E-mail address: akmisra69@gmail.com (A.K. Misra).
Contributed equally.
Figure 1. Structure of the synthesized pentasaccharide and its intermediates.
http://dx.doi.org/10.1016/j.tetasy.2014.03.007
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

M. Jana et al. / Tetrahedron: Asymmetry 25 (2014) 632–636
633
linear synthesis of the pentasaccharide repeating unit of the
O-antigen of E. coli O154 is presented herein (Fig. 1).
by purification using a Sephadex LH-20 column furnished pure
pentasaccharide 1 in 66% overall yield. The structure of compound
1 was confirmed unambiguously from spectroscopic analysis [sig-
nals at d 5.37 (d, J = 3.5 Hz, H-1A), 4.98 (br s, H-1C), 4.89 (br s, H-1D),
4.88 (br s, H-1E), 4.84 (br s, H-1B) in the 1H NMR and at d 102.3
(C-1C), 102.2 (C-1B), 102.1 (C-1D), 97.3 (C-1A), 95.3 (C-1E) in the
13C NMR spectra, respectively] (Scheme 1).
2. Results and discussion
The synthesis of target pentasaccharide 1 was achieved using a
linear synthetic strategy involving sequential glycosylations of suit-
ably functionalized monosaccharide intermediates 2,12 3,13 and 4,14
which were prepared from the reducing sugars using a number of
reported synthetic methodologies. Newly developed glycosylation
conditions using glycosyl trichloroacetimidate derivatives as glyco-
syl donors and nitrosyl tetrafluoroborate (NOBF4) as the glycosyla-
tion activator were used in all of glycosylation reactions throughout
the synthetic scheme.15 General glycosylation conditions were used
in all of the glycosylation reactions and the minimum number of
the protecting group manipulations are involved in the synthetic
strategy. The single monosaccharide intermediate 3 was used sev-
eral times during the progress of oligosaccharide formation, mini-
mizing the overall number of reaction steps.
Ph
O
O
BnO
RO
a
O
B O
A
2 + 3
O
N3
BnO
OPMP
5
6
: R = Ac
: R = H
b
3 a
Stereoselective glycosylation of the 2-azido-
D-galactosyl accep-
Ph
tor 2 with the -rhamnosyl trichloroacetimidate donor 3 in the
L
15
BnO
RO
BnO
O
presence of NOBF4 in Et2O–CH2Cl2 mixed solvent furnished
disaccharide derivative 5 in 78% yield. The formation of compound
5 was confirmed from spectroscopic analysis [signals at d 5.53 (d,
J = 3.5 Hz, H-1A), 5.11 (d, J = 1.5 Hz, H-1B) in the 1H NMR and d
100.8 (C-1B), 97.7 (C-1A) in the 13C NMR spectra, respectively].
Treatment of compound 5 with sodium methoxide16 resulted in
the formation of disaccharide acceptor 6 in 99% yield, which was
coupled with compound 3 in the presence of NOBF415 in Et2O–CH2-
Cl2 mixed solvent to give the trisaccharide derivative 7 in 76%
yield. The stereoselective formation of compound 7 was supported
by spectroscopic analysis [signals at d 5.53 (d, J = 3.0 Hz, H-1A), 5.12
(br s, H-1B), 5.11 (br s, H-1C) in the 1H NMR and at d 100.9 (C-1B),
99.3 (C-1C), 97.5 (C-1A) in the 13C NMR spectra, respectively]. Com-
pound 7 was treated with sodium methoxide16 to give the trisac-
O
O
CO
BO
A
O
O
N3
BnO
BnO
OPMP
7
8
: R = Ac
: R = H
b
3 a
Ph
O
BnO
O
BnO
O
O
BnO
RO
O
B O
C O
DO
A
O
N3
charide acceptor
8 in 97% yield, which was coupled again
BnO
OPMP
BnO
BnO
15
stereoselectively with compound 3 in the presence of NOBF4 in
Et2O–CH2Cl2 mixed solvent to give tetrasaccharide derivative 9 in
74% yield. The formation of compound 9 was confirmed from spec-
troscopic analysis [signals at d 5.51 (d, J = 3.0 Hz, H-1A), 5.14 (br s,
H-1D), 5.12 (br s, H-1B), 5.09 (br s, H-1C) in the 1H NMR and at d
100.7 (C-1B), 99.7 (C-1D), 99.4 (C-1C), 97.5 (C-1A) in the 13C NMR
spectra, respectively]. Treatment of compound 9 with sodium
methoxide16 furnished the tetrasaccharide acceptor 10 in 94%
9
: R = Ac
: R = H
b
10
4
a
Ph
O
N3
BnO
BnO
O
O
BnO
O
AcO
AcO
B O
O
C O
O
E
D O
A
O
O
AcO
N3
yield, which was coupled with 2-azido-D-mannosyl trichloroace-
15
BnO
OPMP
BnO
11
c, d, e
BnO
timidate derivative 4 in the presence of NOBF4 in Et2O–CH2Cl2
mixed solvent to give pentasaccharide derivative 11 in 71% yield.
The formation of compound 11 was confirmed from spectroscopic
analysis [signals at d 5.51 (d, J = 3.0 Hz, H-1A), 5.16 (br s, H-1B), 5.14
(br s, H-1C), 5.13 (br s, H-1D), 4.29 (br s, H-1E) in the 1H NMR
and d 100.6 (JC1–H1 = 171 Hz, C-1B), 99.5 (JC1–H1 = 172 Hz, C-1D),
99.0 (JC1–H1 = 171 Hz, C-1C), 97.5 (JC1–H1 = 171 Hz, C-1A), 93.2
(JC1–H1 = 172 Hz, C-1E) in the 13C NMR, respectively]. The formation
of all of the alpha-glycosyl linkages in compound 11 was also con-
firmed using gated 1H coupled 13C NMR spectra.17 The appearance
1
Scheme 1. Reagents and conditions: (a) NOBF4, Et2O–CH2Cl2 (3:1), ꢀ15 °C, 40 min,
78% for compound 5, 76% for compound 7, 74% for compound 9 and 71% for
compound 11; (b) 0.1 M CH3ONa, CH3OH, room temperature, 1 h, 99% for
compound 6, 97% for compound 8, 94% for compound 10; (c) Et3SiH, 10% Pd-C,
CH3OH/CH2Cl2 (2:1), room temperature, 10 h; (d) acetic anhydride, pyridine, room
temperature, 1 h; (e) 0.1 M CH3ONa, CH3OH, room temperature, 3 h, overall 66%.
of coupling constant (JC1–H1) values 171 Hz, 172 Hz, 171 Hz,
171 Hz, and 172 Hz in the gated 1H coupled 13C NMR spectrum
unambiguously confirmed the presence of five alpha linkages in
compound 11. Removal of the benzyl ethers and benzylidene ace-
tal in compound 11, as well as the reduction of the azido groups,
were carried out using catalytic transfer hydrogenation conditions
by treatment with triethylsilane in the presence of palladium over
charcoal18 to give a pentasaccharide diamine derivative, which was
acetylated using acetic anhydride and pyridine to give a per-O-
acetylated derivative. Finally, saponification of the acetylated
pentasaccharide derivative using sodium methoxide16 followed
3. Conclusion
In conclusion, a pentasaccharide containing all alpha-glycosyl
linkages corresponding to the repeating unit of the O-antigen of
enteroadherent E. coli O154 strain was achieved in good yield using

634
M. Jana et al. / Tetrahedron: Asymmetry 25 (2014) 632–636
similar functional group manipulations and glycosylation condi-
tions involving NOBF4 catalyzed activation of glycosyl trichloroace-
timidate derivatives. The yields were excellent in all of the
glycosylation reactions and intermediate functional group modifi-
cation steps. Multiple uses of a single intermediate and similar
reaction conditions reduced the total number of reaction steps.
The target compound was prepared following a linear glycosyla-
tion approach.
eluant to give pure compound 6 (1.5 g, 99%). Colorless oil;
= +64 (c 1.0, CHCl3); IR (neat): 3434, 3065, 3010, 2930, 2111,
1606, 1507, 1454, 1399, 1367, 1341, 1241, 1215, 1173, 1106,
1083, 1047, 996, 868, 828, 797, 754, 698, 666 cmꢀ1 1H NMR
½ ꢁ
a 2D5
;
(500 MHz, CDCl3): d 7.51–6.81 (m, 19H, Ar-H), 5.54 (s, 1H, PhCH),
5.53 (d, J = 3.5 Hz, 1H, H-1A), 5.16 (br s, 1H, H-1B), 4.88–4.61 (4d,
J = 11.0 Hz each, 4H, 2 PhCH2), 4.43 (d, J = 3.0 Hz, 1H, H-4A), 4.29
(dd, J = 9.5, 3.5 Hz, 1H, H-3A), 4.26–4.24 (m, 1H, H-6aA), 4.09–4.00
(m, 4H, H-2A, H-3B, H-5B, H-6bA), 3.90–3.88 (m, 1H, H-2B), 3.84–
3.83 (m, 1H, H-5A), 3.77 (s, 3H, OCH3), 3.34 (t, J = 9.5 Hz each, 1H,
H-4B), 1.31 (d, J = 6.5 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d
155.6–114.7 (Ar-C), 100.9 (PhCH), 100.1 (C-1B), 97.7 (C-1A), 81.7
(C-4B), 78.7 (C-2B), 76.5 (C-3A), 75.6 (C-4A), 74.3 (PhCH2), 73.3
(PhCH2), 71.3 (C-3B), 69.2 (C-6A), 68.1 (C-5B), 63.3 (C-5A), 58.4 (C-
2A), 55.6 (OCH3), 18.1 (CCH3); ESI-MS: 748.2 [M+Na]+; Anal. Calcd
for C40H43N3O10 (725.29): C, 66.19; H, 5.97; found: C, 66.00; H,
6.12.
4. Experimental
General: All reactions were monitored by thin layer chromatog-
raphy over silica gel coated TLC plates. The spots on TLC were visu-
alized by warming ceric sulfate (2% Ce(SO4)2 in 2 M H2SO4) sprayed
plates in hot plate. Silica gel 230–400 mesh was used for column
chromatography. 1H and 13C NMR spectra were recorded on Bruc-
ker Avance 500 MHz using CDCl3 and D2O as the solvents and TMS
as the internal reference unless stated otherwise. Chemical shift
values are expressed in d ppm. MALDI-MS were recorded on a
Brucker Daltronics mass spectrometer. Optical rotations were re-
corded in a Jasco P-2000 spectrometer. Commercially available
grades of organic solvents of adequate purity are used in all
reactions.
4.3. p-Methoxyphenyl (3-O-acetyl-2,4-di-O-benzyl-
pyranosyl)-(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-
2-azido-4,6-O-benzylidene-2-deoxy- -galactopyranoside 7
a-L-rhamno-
a
-L
a
-D
A solution of compound 6 (1.4 g, 1.93 mmol) and compound 3
(1.1 g, 2.07 mmol) in anhydrous Et2O–CH2Cl2 (15 mL; 3:1; v/v)
was cooled to ꢀ15 °C under argon and NOBF4 (260 mg, 2.22 mmol)
was added to it. The reaction mixture was allowed to stir at same
temperature for 40 min and poured into a satd. NaHCO3 solution
and extracted with CH2Cl2 (100 mL). The organic layer was dried
over Na2SO4 and concentrated under reduced pressure to give
the crude product, which was purified over SiO2 using hexane–
EtOAc (6:1) as eluant to give pure compound 7 (1.6 g, 76%). Color-
4.1. p-Methoxyphenyl (3-O-acetyl-2,4-di-O-benzyl-
pyranosyl)-(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-a-D
a
-
L
-rhamno-
-gala-
ctopyranoside 5
A solution of compound 2 (1.2 g, 3.0 mmol) and compound 3
(1.7 g, 3.20 mmol) in anhydrous Et2O–CH2Cl2 (15 mL; 3:1; v/v)
was cooled to ꢀ15 °C under argon and NOBF4 (400 mg, 3.42 mmol)
was added to it. The reaction mixture was allowed to stir at the
same temperature for 40 min and poured into a satd. NaHCO3 solu-
tion and extracted with CH2Cl2 (100 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure to
give the crude product, which was purified over SiO2 using hex-
ane–EtOAc (5:1) as eluant to give pure compound 5 (1.8 g, 78%).
less oil; ½a 2D5
ꢁ
= +54 (c 1.0, CHCl3); IR (neat): 3367, 3064, 3030, 2931,
2112, 1735, 1507, 1454, 1365, 1342, 1240, 1216, 1173, 1103, 1083,
1048, 999, 916, 893, 828, 796, 755, 698, 667 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d 7.55–6.81 (m, 29H, Ar-H), 5.55 (s, 1H, PhCH),
5.53 (d, J = 3.0 Hz, 1H, H-1A), 5.27 (dd, J = 9.5, 3.0 Hz, 1H, H-3C),
5.12 (br s, 1H, H-1B), 5.11 (br s, 1H, H-1C), 4.82–4.58 (6 d,
J = 11.0 Hz each, 6H, PhCH2), 4.47 (d, J = 2.5 Hz, 1H, H-4A), 4.30
(dd, J = 9.5, 3.0 Hz, 1H, H-3A), 4.27–4.24 (m, 2H, H-6aA, PhCH2),
4.15 (dd, J = 9.5, 3.0 Hz, 1H, H-3B), 4.12–4.02 (m, 5H, H-2A, H-5B,
H-5C, H-6bA, PhCH2), 3.91–3.89 (m, 1H, H-2B), 3.84–3.83 (m, 1H,
H-5A), 3.80–3.78 (m, 1H, H-2C), 3.77 (s, 3H, OCH3), 3.63 (t,
J = 9.5 Hz each, 1H, H-4C), 3.59 (t, J = 9.5 Hz each, 1H, H-4B), 1.92
(s, 3H, COCH3), 1.26 (d, J = 6.5 Hz, 3H, CCH3), 1.22 (d, J = 6.5 Hz,
3H, CCH3); 13C NMR (125 MHz, CDCl3): d 170.1 (COCH3), 156.2–
114.7 (Ar-C), 101.0 (PhCH), 100.9 (C-1B), 99.3 (C-1C), 97.5 (C-1A),
80.5 (C-4B), 79.1 (C-4C), 77.9 (C-2B), 77.4 (C-2C), 76.9 (2 C, C-3A,
C-3B), 75.7 (C-4A), 74.7 (PhCH2), 74.2 (PhCH2), 73.5 (C-3C), 73.1
(PhCH2), 72.4 (PhCH2), 69.2 (C-6A), 69.0 (C-5C), 68.4 (C-5B), 63.2
(C-5A), 58.3 (C-2A), 55.6 (OCH3), 21.0 (COCH3), 18.0, 17.9 (2CCH3);
MALDI-MS: 1116.4 [M+Na]+; Anal. Calcd for C62H67N3O15
(1093.45): C, 68.06; H, 6.17; found: C, 67.90; H, 6.32.
Colorless oil; ½a D25
ꢁ
= +49 (c 1.0, CHCl3); IR (neat): 3400, 3065,
3030, 2921, 2111, 1738, 1594, 1506, 1486, 1454, 1367, 1239,
1103, 1083, 1047, 999, 917, 893, 867, 828, 797, 755, 698,
666 cmꢀ1 1H NMR (500 MHz, CDCl3): d 7.52–6.81 (m, 19H, Ar-
;
H), 5.54 (s, 1H, PhCH), 5.53 (d, J = 3.5 Hz, 1H, H-1A), 5.24 (dd,
J = 9.5, 3.5 Hz, 1H, H-3B), 5.11 (d, J = 1.5 Hz, 1H, H-1B), 4.70–4.57
(4d, J = 11.5 Hz each, 4H, 2PhCH2), 4.45 (d, J = 3.0 Hz, 1H, H-4A),
4.31 (dd, J = 9.5, 3.5 Hz, 1H, H-3A), 4.26–4.24 (m, 1H, H-6aA),
4.18–4.12 (m, 1H, H-5B), 4.09 (dd, J = 9.5, 3.5 Hz, 1H, H-2A), 4.05–
4.01 (m, 2H, H-2B, H-6bA), 3.83 (br s, 1H, H-5A), 3.77 (s, 3H,
OCH3), 3.61 (t, J = 9.5 Hz each, 1H, H-4B), 1.92 (s, 3H, COCH3),
1.29 (d, J = 6.5 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d 169.8
(COCH3), 156.4–114.7 (Ar-C), 100.9 (PhCH), 100.8 (C-1B), 97.7 (C-
1A), 78.8 (C-4B), 76.9 (C-3A), 76.3 (C-4A), 75.6 (C-2B), 74.0 (PhCH2),
73.3 (PhCH2), 73.1 (C-3B), 69.2 (C-6A), 68.4 (C-5B), 63.3 (C-5A), 58.2
(C-2A), 55.6 (OCH3), 21.0 (COCH3), 18.1 (CCH3); ESI-MS: 790.3
[M+Na]+; Anal. Calcd for C42H45N3O11 (767.30): C, 65.70; H, 5.91;
found: C, 65.24; H, 6.10.
4.4. p-Methoxyphenyl (2,4-di-O-benzyl-
(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-2-azido-
4,6-O-benzylidene-2-deoxy- -galactopyranoside 8
a-L-rhamnopyranosyl)-
a-L
a
-D
4.2. p-Methoxyphenyl (2,4-di-O-benzyl-
(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-a-D
side 6
a
-
L
-rhamnopyranosyl)-
-galactopyrano-
A solution of compound 7 (1.5 g, 1.37 mmol) in 0.1 M CH3ONa
in CH3OH (20 mL) was allowed to stir at room temperature for
1 h. The reaction mixture was neutralized with Dowex 50W-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 8 (1.4 g, 97%). Colorless oil;
A solution of compound 5 (1.6 g, 2.08 mmol) in 0.1 M CH3ONa
in CH3OH (20 mL) was allowed to stir at room temperature for
1 h. The reaction mixture was neutralized with Dowex 50W-X8
(H+) resin, filtered, and concentrated. The crude product was
passed through a small pad of SiO2 using hexane-EtOAc (1:1) as
½
a 2D5
ꢁ
= +55 (c 1.0, CHCl3); IR (neat): 3400, 3031, 2931, 2111, 1594,
1507, 1454, 1396, 1341, 1240, 1214, 1172, 1083, 1048, 998, 918,

M. Jana et al. / Tetrahedron: Asymmetry 25 (2014) 632–636
635
828, 753, 697 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d 7.55-6.81 (m,
eluant to give pure compound 10 (1 g, 94%). Colorless oil;
= +115 (c 1.0, CHCl3); IR (neat): 3551, 3088, 3064, 3030,
29H, Ar-H), 5.55 (s, 1H, PhCH), 5.52 (d, J = 3.0 Hz, 1H, H-1A), 5.15
(br s, 1H, H-1B), 5.10 (br s, 1H, H-1C), 4.88–4.59 (6d, J = 11.0 Hz
each, 6H, PhCH2), 4.47 (d, J = 3.0 Hz, 1H, H-4A), 4.28 (dd, J = 9.5,
3.0 Hz, 1H, H-3A), 4.26–4.20 (m, 2H, H-6aA, PhCH2), 4.17 (dd,
J = 9.5, 3.0 Hz, 1H, H-3B), 4.15–4.09 (m, 2H, H-2A, H-3C), 4.05 (d,
J = 11.5 Hz, 1H, PhCH2), 3.98–3.91 (m, 2H, H-5B, H-6bA), 3.90–3.89
(m, 1H, H-2B), 3.84–3.83 (m, 1H, H-5A), 3.77 (s, 3H, OCH3), 3.75–
3.70 (m, 1H, H-5C), 3.65–3.63 (m, 1H, H-2C), 3.61 (t, J = 9.5 Hz each,
1H, H-4C), 3.25 (t, J = 9.5 Hz each, 1H, H-4B), 1.28 (d, J = 6.5 Hz, 3H,
CCH3), 1.22 (d, J = 6.5 Hz, 3H, CCH3); 13C NMR (125 MHz, CDCl3): d
156.2–114.7 (Ar-C), 101.0 (PhCH), 100.8 (C-1B), 98.7 (C-1C), 97.5
(C-1A), 82.2 (C-4B), 80.7 (C-4C), 79.2 (C-2B), 78.0 (C-2C), 77.4 (C-
3A), 76.9 (C-3B), 75.7 (C-4A), 74.9 (PhCH2), 74.2 (PhCH2), 73.1
(PhCH2), 72.3 (PhCH2), 71.5 (C-3C), 69.2 (C-6A), 69.0 (C-5C), 67.8
(C-5B), 63.2 (C-5A), 58.3 (C-2A), 55.6 (OCH3), 18.0, 17.9 (2CCH3);
MALDI-MS: 1074.4 [M+Na]+; Anal. Calcd for C60H65N3O14
(1051.44): C, 68.49; H, 6.23; found: C, 68.32; H, 6.42.
½ ꢁ
a 2D5
2972, 2932, 2112, 1953, 1877, 1811, 1729, 1596, 1486, 1454,
1394, 1366, 1342, 1316, 1269, 1214, 1173, 1083, 1048, 999, 918,
837, 803, 754, 697, 667, 618, 586 cmꢀ1; 1H NMR (500 MHz, CDCl3):
d 7.55–6.74 (m, 39H, Ar-H), 5.54 (s, 1H, PhCH), 5.51 (d, J = 3.0 Hz,
1H, H-1A), 5.14–5.12 (3br s, 3H, H-1B, H-1C, H-1D), 4.86–4.58 (8 d,
J = 11.0 Hz each, 8H, PhCH2), 4.46 (d, J = 2.5 Hz, 1H, H-4A), 4.30–
4.22 (m, 5H, H-3A, H-3B, H-3C, PhCH2), 4.15–4.02 (m, 7H, H-2A, H-
3D, H-5B, H-6abA, PhCH2), 3.97–3.85 (m, 2H, H-2B, H-3D), 3.83 (s,
3H, OCH3), 3.80–3.79 (m, 1H, H-2D), 3.78–3.77 (m, 1H, H-5A),
3.76–3.70 (m, 2H, H-5C, H-5D), 3.64–3.55 (m, 3H, H-2C, H-4B, H-
4C), 3.26 (t, J = 9.5 Hz each, 1H, H-4D), 1.26–1.18 (3 d, J = 6.5 Hz
each, 9H, 3CCH3); 13C NMR (125 MHz, CDCl3): d 155.6–111.2 (Ar-
C), 101.0 (PhCH), 100.7 (C-1B), 99.4 (C-1D), 98.9 (C-1C), 97.5 (C-
1A), 82.2 (C-4D), 80.8 (C-4B), 80.4 (C-4C), 79.1 (2C, C-2B, C-2C),
78.6 (C-2D), 78.4 (C-3D), 78.0 (C-3A), 77.4 (C-4A), 75.5 (C-3B), 74.6
(PhCH2), 74.5 (PhCH2), 74.3 (PhCH2), 73.0 (PhCH2), 72.4 (PhCH2),
72.1 (PhCH2), 71.5 (C-3C), 69.2 (C-6A), 69.0 (C-5D), 68.9 (C-5C),
67.6 (C-5B), 63.4 (C-5A), 58.2 (C-2A), 56.8 (OCH3), 18.1, 18.0, 17.9
(3CCH3); MALDI-MS: 1400.5 [M+Na]+; Anal. Calcd for
4.5. p-Methoxyphenyl (3-O-acetyl-2,4-di-O-benzyl-
pyranosyl)-(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-
(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-2-azido-4,6-O-
benzylidene-2-deoxy- -galactopyranoside 9
a-L-rhamno-
a-L
a
-
L
C80H87N3O18 (1377.59): C, 69.70; H, 6.36; found: C, 69.53; H, 6.54.
a-D
4.7. p-Methoxyphenyl (3,4,6-tri-O-acetyl-2-azido-2-deoxy-
mannopyranosyl)-(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-
(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-(2,4-
di-O-benzyl- -rhamnopyranosyl)-(1?3)-2-azido-4,6-O-benzyl-
idene-2-deoxy- -galactopyranoside 11
a-D-
A solution of compound 8 (1.2 g, 1.14 mmol) and compound 3
(670 mg, 1.26 mmol) in anhydrous Et2O–CH2Cl2 (10 mL; 3:1; v/v)
was cooled to ꢀ15 °C under argon and NOBF4 (160 mg, 1.37 mmol)
was added to it. The reaction mixture was allowed to stir at same
temperature for 40 min and then poured into a satd. NaHCO3 solu-
tion and extracted with CH2Cl2 (100 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure to
give the crude product, which was purified over SiO2 using hex-
ane–EtOAc (6:1) as eluant to give pure compound 9 (1.2 g, 74%).
a-L
a
-L
a
-L
a
-D
A solution of compound 10 (900 mg, 0.65 mmol) and compound
4 (340 mg, 0.71 mmol) in anhydrous Et2O–CH2Cl2 (10 mL; 3:1; v/v)
was cooled to ꢀ15 °C under argon and NOBF4 (90 mg, 0.77 mmol)
was added to it. The reaction mixture was allowed to stir at same
temperature for 40 min and then poured into a satd. NaHCO3 solu-
tion and extracted with CH2Cl2 (100 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure to
give the crude product, which was purified over SiO2 using hex-
ane–EtOAc (5:1) as eluant to give pure compound 11 (780 mg,
Colorless oil; ½a D25
ꢁ
= +71 (c 1.0, CHCl3); IR (neat): 3368, 3030,
2926, 2112, 1737, 1596, 1486, 1454, 1366, 1342, 1239, 1214,
1101, 1048, 999, 918, 804, 755, 697, 666 cmꢀ1
;
1H NMR
(500 MHz, CDCl3): d 7.56–6.75 (m, 39H, Ar-H), 5.54 (s, 1H, PhCH),
5.51 (d, J = 3.0 Hz, 1H, H-1A), 5.25 (dd, J = 9.5, 3.0 Hz, 1H, H-3D),
5.14 (br s, 1H, H-1D), 5.12 (br s, 1H, H-1B), 5.09 (br s, 1H, H-1C),
4.80–4.57 (8 d, J = 11.0 Hz each, 8H, PhCH2), 4.46 (d, J = 2.5 Hz,
1H, H-4A), 4.32–4.24 (m, 5H, H-3A, H-3B, H-3C, PhCH2), 4.15–4.09
(m, 5H, H-2A, H-5B, H-6aA, PhCH2), 4.02 (d, J = 12.0 Hz, 1H, H-6bA),
3.92–3.91 (m, 1H, H-2B), 3.84 (s, 3H, OCH3), 3.82–3.77 (m, 4H, H-
2D, H-5A, H-5C, H-5D), 3.74–3.73 (m, 1H, H-2C), 3.60–3.56 (m, 3H,
H-4B, H-4C, H-4D), 1.91 (s, 3H, COCH3), 1.26, 1.20, 1.18 (3 d,
J = 6.5 Hz each, 9H, 3CCH3); 13C NMR (125 MHz, CDCl3): d 169.9
(COCH3), 155.2–111.2 (Ar-C), 101.0 (PhCH), 100.7 (C-1B), 99.7 (C-
1D), 99.4 (C-1C), 97.5 (C-1A), 80.6 (C-4B), 80.5 (C-4C), 79.0 (C-4D),
78.6 (2 C, C-2B, C-2C), 78.3 (C-2D), 78.0 (C-3D), 77.4 (C-3A), 76.7
(C-3B), 75.5 (C-4A), 74.6 (PhCH2), 74.5 (PhCH2), 74.3 (PhCH2), 73.5
(C-3C), 73.0 (PhCH2), 72.6 (PhCH2), 72.1 (PhCH2), 69.1 (C-6A), 69.0
(C-5D), 68.9 (C-5C), 68.2 (C-5B), 63.4 (C-5A), 58.2 (C-2A), 56.8
(OCH3), 21.0 (COCH3), 18.1, 18.0, 17.8 (3CCH3); MALDI-MS:
1442.4 [M+Na]+; Anal. Calcd for C82H89N3O19 (1419.60): C, 69.33;
H, 6.31; found: C, 69.15; H, 6.50.
71%). Colorless oil; ½a D25
ꢁ
= +44 (c 1.0, CHCl3); IR (neat): 3368,
2927, 2110, 1748, 1587, 1507, 1454, 1367, 1219, 1080, 1047,
918, 828, 752, 697 cmꢀ1 1H NMR (500 MHz, CDCl3): d 7.55–6.78
;
(m, 39H, Ar-H), 5.55 (s, 1H, PhCH), 5.51 (d, J = 3.0 Hz, 1H, H-1A),
5.28 (dd, J = 9.5, 3.0 Hz, 1H, H-3E), 5.18 (t, J = 9.5 Hz each, 1H, H-
4E), 5.16 (br s, 1H, H-1B), 5.14 (br s, 1H, H-1C), 5.13 (br s, 1H, H-
1D), 4.85–4.63 (8 d, J = 11.0 Hz each, 8H, PhCH2), 4.47 (d,
J = 11.0 Hz, 1H, PhCH2), 4.46 (d, J = 2.5 Hz, 1H, H-4A), 4.29 (br s,
1H, H-1E), 4.28–4.22 (m, 3H, H-3A, PhCH2), 4.18–4.07 (m, 5H, H-
2A, H-3B, H-5B, H-6aA, PhCH2), 4.05 (d, J = 12.0 Hz, 1H, H-6bA),
4.00–3.92 (m, 4H, H-2B, H-2C, H-3C, H-3D), 3.87–3.84 (m, 1H, H-
6aE), 3.83 (s, 3H, OCH3), 3.81–3.78 (m, 3H, H-5A, H-5C, H-5E),
3.75–3.72 (m, 1H, H-5D), 3.65–3.59 (m, 3H, H-2D, H-4B, H-6bE),
3.52 (t, J = 9.5 Hz each, 1H, H-4C), 3.50 (t, J = 9.5 Hz each, 1H, H-
4D), 3.46–3.44 (m, 1H, H-2E), 2.09, 1.86, 1.83 (3s, 9H, 3COCH3),
1.27, 1.23, 1.21 (3 d, J = 6.5 Hz each, 9H, 3CCH3); 13C NMR
(125 MHz, CDCl3): d 170.6, 170.2, 170.0 (COCH3), 156.2–111.2
(Ar-C), 101.0 (PhCH), 100.6 (JC1–H1 = 171 Hz, C-1B), 99.5
(JC1–H1 = 172 Hz, C-1D), 99.0 (JC1–H1 = 171 Hz, C-1C), 97.5
(JC1–H1 = 171 Hz, C-1A), 93.2 (JC1–H1 = 172 Hz, C-1E), 80.4 (3 C,
C-4B, C-4C, C-4D), 79.4 (C-2B), 79.0 (C-2C), 78.5 (C-2D), 78.4 (C-3D),
77.5 (C-3A), 75.5 (2C, C-3B, C-4A), 75.1 (PhCH2), 74.5 (PhCH2), 74.3
(PhCH2), 73.6 (C-3C), 73.0 (2C, C-3C, PhCH2), 72.1 (PhCH2), 71.9
(PhCH2), 71.2 (C-3E), 69.1 (C-6A), 69.0 (C-5E), 68.8 (C-5D), 68.5 (C-
5C), 68.1 (C-5B), 65.3 (C-4E), 63.4 (C-5A), 61.3 (C-6E), 61.2 (C-2E),
58.2 (C-2A), 56.8 (OCH3), 20.5 (3C, 3COCH3), 18.1, 18.0, 17.9
(3CCH3); MALDI-MS: 1713.6 [M+Na]+; Anal. Calcd for
4.6. p-Methoxyphenyl (2,4-di-O-benzyl-
(1?3)-(2,4-di-O-benzyl- -rhamnopyranosyl)-(1?3)-(2,4-di-
O-benzyl- -rhamnopyranosyl)-(1?3)-2-azido-4,6-O-benzyl-
idene-2-deoxy- -galactopyranoside 10
a-L-rhamnopyranosyl)-
a
-L
a
-L
a-L
A solution of compound 9 (1.1 g, 0.77 mmol) in 0.1 M CH3ONa
in CH3OH (15 mL) was allowed to stir at room temperature for
1 h. The reaction mixture was neutralized with Dowex 50W-X8
(H+) resin, filtered, and concentrated. The crude product was
passed through a small pad of SiO2 using hexane–EtOAc (1:1) as
C92H102N6O25 (1690.68): C, 65.31; H, 6.08; found: C, 65.15; H, 6.30.

636
M. Jana et al. / Tetrahedron: Asymmetry 25 (2014) 632–636
4.8. p-Methoxyphenyl (2-acetamido-2-deoxy-
a
-
D
-mannopyran-
-rhamnopyrano-
-rhamnopyranosyl)-(1?3)-2-acetamido-2-deoxy-
-D-galactopyranoside 1
(C-4D), 71.2 (C-2C), 70.3 (C-4C), 70.2 (C-2B), 69.9 (C-4B), 69.4 (C-
3E), 69.2 (C-5B), 69.1 (C-5D), 68.9 (C-5C), 68.4 (C-4A), 66.4 (C-4E),
66.1 (C-2D), 60.9 (C-6A), 60.2 (C-6E), 55.8 (OCH3), 52.8 (C-2E), 48.5
(C-2A), 21.9, 21.8 (2COCH3), 16.7 (3C, 3CCH3); ESI-MS: 991.3
[M+Na]+; Anal. Calcd for C41H64N2O24 (968.38): C, 50.82; H, 6.66;
found: C, 50.65; H, 6.40.
osyl)-(1?3)-(a-L-rhamnopyranosyl)-(1?3)-(a-L
syl)-(1?3)-( -L
a
a
To a solution of compound 11 (700 mg, 0.41 mmol) and 10% Pd-
C (150 mg) in CH3OH–CH2Cl2 (10 mL, 2:1; v/v) was added dropwise
Et3SiH (3 mL, 18.78 mmol) over 30 min and the reaction mixture
was allowed to stir at room temperature for 10 h. The reaction
mixture was then filtered through a CeliteÒ bed, washed with
CH3OH (50 mL), and the combined filtrate was concentrated under
reduced pressure. A solution of the crude product in acetic anhy-
dride (5 mL) and pyridine (5 mL) was kept at room temperature for
1 h and evaporated to dryness under reduced pressure. A solution
of the acetylated product in 0.1 M CH3ONa (15 mL) was stirred at
room temperature for 3 h. The reaction mixture was neutralized
using Dowex 50W X8 (H+) resin, filtered, and concentrated to give
the product, which was passed through a SephadexÒ LH-20 column
using CH3OH–H2O (3:1) as the eluent to give pure compound 1
Acknowledgements
M.J. and A.S. thank CSIR, New Delhi for providing Senior Re-
search Fellowships. This work was supported by DST, New Delhi
(No. SR/S1/OC-83/2010).
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ꢁ
= 130 (c 1.0, H2O); IR (KBr):
3434, 2936, 2851, 1721, 1657, 1590, 1400, 1056, 996 cmꢀ1
;
1H
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J = 9.0 Hz, 2H, Ar-H), 5.37 (d, J = 3.5 Hz, 1H, H-1A), 4.98 (br s, 1H,
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H-3A, H-3E, H-4A), 3.89–3.85 (m, 1H, H-5E), 3.82–3.74 (m, 7H,
H-3B, H-3C, H-3D, H-5A, H-5B, H-5C, H-5D), 3.72–3.70 (m, 2H, H-6abA),
3.68 (s, 3H, OCH3), 3.62–3.59 (m, 2H, H-6abE), 3.57 (t, J = 9.5 Hz
each, 1H, H-4E), 3.47–3.42 (m, 3H, H-4B, H-4C, H-4D), 1.96, 1.95
(2s, 6H, 2 COCH3), 1.20–1.19 (m, 9H, 3CCH3); 13C NMR (125 MHz,
D2O): d 174.8, 174.2 (2COCH3), 154.6–115.0 (Ar-C), 102.3 (C-1C),
102.2 (C-1B), 102.1 (C-1D), 97.3 (C-1A), 95.3 (C-1E), 78.4 (C-3C),
77.9 (C-3B), 76.7 (C-3A), 74.5 (C-3D), 72.2 (C-5E), 71.7 (C-5A), 71.3
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