Synthesis of a di- and a trisaccharide related to the O-antigen of Escherichia coli O83:K24:H31

Article abstract of DOI:10.1016/j.carres.2006.07.007

Di- and trisaccharide fragments related to the repeating unit of the O-antigen of Escherichia coli O83:K24:H31 have been synthesized as their methyl glycoside analogs starting from readily available monosaccharides.

Full text of DOI:10.1016/j.carres.2006.07.007

Carbohydrate Research 341 (2006) 2420–2425
Note
Synthesis of a di- and a trisaccharide related to the O-antigen of
Escherichia coli O83:K24:H31I
Geetanjali Agnihotri and Anup Kumar Misra*
Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001, UP, India
Received 7 June 2006; received in revised form 6 July 2006; accepted 13 July 2006
Available online 1 August 2006
Abstract—Di- and trisaccharide fragments related to the repeating unit of the O-antigen of Escherichia coli O83:K24:H31 have been
synthesized as their methyl glycoside analogs starting from readily available monosaccharides.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Colinfant; Vaccine; Glycosylation; E. coli; Disaccharide; Trisaccharide
‘Colinfant’, a commercially used oligosaccharide vac-
cine, contains the bacterial strain Escherichia coli
O83:K24:H31.1 In the gut, the bacteria stimulates the
production of specific and nonspecific antibodies both
at the local level within the gut and in the saliva as well
as in the blood. The ‘Colinfant’ oligosaccharide vaccine
decreased the number of nosocomial infections, mortal-
ity rate in connection with infection, and the need for
antibiotics.2,3 The structure of the O-antigen of E. coli
O83:K24:H31 has been established.4 In the light of
increasing drug resistance of pathogenic bacterial infec-
tions and the potential importance of artificial antigens,
the selective chemical synthesis of immunodominant
fragments of bacterial O-antigens, which could be used
to study the potential of synthetic antigens for diagnosis
and protection, has gained considerable interest. To
understand the structure–activity relationship and anti-
genic properties of the pentasaccharide repeating unit
of this polysaccharide, b-D-GlcpA-(1!6)-b-D-Galp-
(1!4)-b-D-Galp-(1!4)-b-D-GlcpNAc-(1!6)-a-D-Glcp-,
we prepared di- and trisaccharide fragments of it,
namely b-D-GlcpA-(1!6)-b-D-Galp-OCH3 and b-D-
Galp-(1!4)-b-D-GlcpNAc-(1!6)-a-D-Glcp-OCH3.
Recently, perchloric acid supported on silica (HClO4–
SiO2) has been successfully used by us and others in sev-
eral carbohydrate transformations and glycosylation
reactions.5,6 As a part of our continuing efforts to syn-
thesize biologically important carbohydrate haptens in
a concise manner, we disclose here a concise approach
for the synthesis of the aforementioned di- and trisac-
charides using HClO4–SiO2-mediated transformations.
The synthesis of the disaccharide fragment is shown in
Scheme 1. Methyl 2,3,4-tri-O-acetyl-b-D-galactopyran-
oside (2) was prepared from methyl b-D-galactopyran-
oside (1) in three steps involving conventional
tritylation,7 acetylation, and de-tritylation using
8
HClO4–SiO2 in 85% overall yield. Initial attempts to
glycosylate 2 with methyl (ethyl 2,3,4-tri-O-acetyl-
l-thio-b-D-glucopyranoside)uronate (3) using either N-
6c
iodosuccinimide (NIS) and HClO4–SiO2 or NIS–
TfOH did not result in any glycosylated product, which
may be explained due to the poor reactivity of glucu-
ronic acid donor. Therefore, following the classical ap-
proach, glycosylation of 2 with methyl (2,3,4-tri-O-
acetyl-a-D-glucopyranosyl bromide)uronate (4)9 using
silver trifluoromethanesulfonate as the activator10 was
carried out, which yielded the expected product, 5, in
85% yield. Deacetylation followed by hydrolysis of the
methyl ester in 5 using 0.05 M sodium methoxide and
a few drops of water furnished the target disaccharide
6 in 90% yield. The presence of two doublets at d 4.82
q CDRI Communication No. 6971.
*
Corresponding author. Tel.: +91 522262411; fax: +91 5222623405;
e-mail: akmisra69@rediffmail.com
0008-6215/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.07.007

G. Agnihotri, A. K. Misra / Carbohydrate Research 341 (2006) 2420–2425
2421
COOMe
O
AcO
AcO
COONa
O
AcO
COOMe
4
c
Br
HO
HO
O
AcO
AcO
O
HO
HO
HO
OH
AcO
AcO
OH
O
d
COOMe
HO
O
AcO
a
O
AcO
O
O
AcO
AcO
OMe
OMe
O
SEt
OH
OMe
HO
OAc
6
OMe
AcO
AcO
3
2
5
OH
1
OAc
x
b
Scheme 1. Reagents and conditions: (a) (1) TrCl, pyridine, rt, 48 h; (2) Ac2O, pyridine, 0 °C, 5 h; (3) HClO4–SiO2, rt, 1 h; 85% in three steps; (b) NIS,
˚
˚
HClO4–SiO2, 4 A molecular sieves, CH2Cl2, 0 °C, 1 h; (c) AgOTf, 4 A molecular sieves, CH2Cl2, ꢀ20 °C, 4 h, 85%; (d) 0.05 M NaOCH3, CH3OH, rt,
6 h, then a few drops of water, rt, 12 h, 90%.
(J = 8.1 Hz) and 4.44 (J = 8.1 Hz) in the 1H NMR spec-
trum and two anomeric signals at d 102.9 (C-1) and
101.6 (C-10) in the 13C NMR spectrum of 6 confirmed
the structure of the product.
First, the benzylidene acetal was removed using HClO4–
SiO2 to give disaccharide diol 12 and then selective
benzoylation13 of the primary hydroxyl using benzoyl
cyanide afforded 13. Glycosylation of 13 with thiogly-
coside 1414 using NIS and HClO4–SiO2 furnished the
trisaccharide derivative 15 in 82% yield. Signals at d
5.48 (d, J = 8.4 Hz), 5.28 (d, J = 10.2 Hz) and 4.44 (d,
The trisaccharide target was prepared as illustrated in
Scheme 2. Thus, methyl 2,3,4-tri-O-acetyl-a-D-glucopyr-
anoside (8)11 was prepared from methyl a-D-glucopyr-
anoside (7) in three steps: tritylation, acetylation, and
de-tritylation in 80% overall yield. Glycosylation of 8
with phenyl thioglycoside 10,12 which was prepared
1
J = 3.3 Hz) in the H NMR and at d 98.4 (C-10) and
96.1 (C-100 and C-1) in 13C NMR spectra of 15 con-
firmed the formation of desired trisaccharide derivative.
Removal of the phthalimido group using hydrazine
hydrate15 followed by N-acetylation and global deacetyl-
ation afforded the target trisaccharide 16 in 70% yield
6c
from 9 in four steps, using NIS and HClO4–SiO2 pro-
vided disaccharide 11 in 80% yield. This product was
transformed into disaccharide acceptor 13 in two steps.
OH
OH
O
HO
HO
a
O
AcO
AcO
Ph
O
HO
O
N
O
AcO
OMe
O
7
AcO
OMe
d
8
c
O
O
O
AcO
OAc
Ph
O
AcO
O
N
b
O
O
AcO
AcO
AcO
SPh
AcO
OAc
O
OMe
11
N
O
O
O
10
9
OH
OBz
AcO
AcO
OAc
HO
AcO
O
HO
O
O
O
O
AcO
SPh
e
N
N
OAc
O
O
O
O
O
AcO
AcO
O
AcO
AcO
14
f
AcO
AcO
13
OMe
12
OMe
O
AcO
AcO
OAc
HO
HO
OH
OBz
O
OH
O
O
O
O
HO
g
AcO
O
AcO
OH
O
N
O
O
O
NHAc
HO
HO
AcO
AcO
O
AcO
16
15
HO
OMe
OMe
Scheme 2. Reagents and conditions: (a) (1) TrCl, pyridine, rt, 48 h; (2) Ac2O, pyridine, 0 °C, 5 h; (3) HClO4–SiO2, rt, 1 h, 80% in three steps; (b) (1)
PhSH, BF3ÆOEt2, CH2Cl2, 0 °C–rt, 5 h, 90%; (2) 0.05 M NaOCH3, CH3OH, rt, 20 min, quantitative; (3) PhCH(OCH3)2, HClO4–SiO2, CH3CN, rt,
˚
2 h, then Ac2O, 0 °C, 1 h, 85% in two steps; (c) NIS, HClO4–SiO2, 4 A molecular sieves, CH2Cl2, 0 °C, 1.5 h, 80%; (d) HClO4–SiO2, CH3CN, rt,
˚
30 min, 95%; (e) BzCN, pyridine, CH2Cl2, rt, 12 h, 90%; (f) NIS, HClO4–SiO2, 4 A molecular sieves, CH2Cl2, 0 °C, 2 h, 82%; (g) (1) NH2NH2ÆH2O,
EtOH, 80 °C, 8 h, (2) Ac2O, pyridine, rt, 2 h; (3) 0.05 M NaOCH3, CH3OH, rt, 12 h, 70% in three steps.

2422
G. Agnihotri, A. K. Misra / Carbohydrate Research 341 (2006) 2420–2425
(Scheme 2). The structure of 16 was confirmed by NMR
and mass spectroscopy.
MHz, CDCl3): d 5.38 (d, J = 3.3 Hz, 1H, H-4), 5.22
(dd, J = 8.1 and 10.5 Hz, 1H, H-2), 5.04 (dd, J = 3.3
and 10.5 Hz, 1H, H-3), 4.41 (d, J = 7.8 Hz, 1H, H-1),
3.79–3.71 (m, 2H, H-6ab), 3.59–3.54 (m, 1H, H-5), 3.52
(s, 3H, OCH3), 2.45–2.39 (br s, 1H, OH), 2.17, 2.08,
1.99 (3s, 9H, 3 COCH3); 13C NMR (75 MHz, CDCl3):
d 171.1, 170.0, 169.5, 102.1 (C-1), 73.47, 71.0, 69.1,
67.9, 60.5, 56.9 (OCH3), 20.68, 20.59, 20.49; ESIMS:
m/z 343.1 [M+Na]+. Anal. Calcd for C13H20O9: C,
48.75; H, 6.29. Found: C, 48.58; H, 6.50.
In summary, we have developed a concise synthesis of
di- and trisaccharide methyl glycoside fragments of the
pentasaccharide repeating unit of the O-antigen of
E. coli O83:K24:H31, which is present in the carbohy-
drate vaccine ‘Colinfant’.
1. Experimental
1.1. General experimental methods
1.4. Methyl (methyl 2,3,4-tri-O-acetyl-b-D-glucopyran-
osyl)uronate-(1!6)-2,3,4-tri-O-acetyl-b-D-galactopyran-
oside (5)
All reactions were monitored by thin layer chromatogra-
phy on 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 on a hot plate. Silica gel
230–400 mesh was used for column chromatography.
1H and 13C NMR spectra were recorded on a Bruker
Advance DPX 300 MHz using TMS as the internal ref-
erence. Chemical shift values are expressed in ppm. Ele-
mental analysis was carried out on Carlo ERBA-1108
analyzer. Optical rotations were measured at 25 °C on
a Rudolf Autopol III polarimeter. Commercially avail-
able grades of organic solvents of adequate purity are
used in the reactions.
To a solution of 2 (650.0 mg, 2.03 mmol) in CH2Cl2
˚
(10.0 mL) were added 4 A molecular sieves (1.0 g) and
silver trifluoromethanesulfonate (1.2 g, 4.67 mmol) and
the mixture was stirred under argon at ꢀ20 °C for
30 min. A solution of 4 (1.40 g, 3.5 mmol) in CH2Cl2
(5.0 mL) was added and the mixture was stirred at
ꢀ20 °C for 4 h, before being filtered and washed with
CH2Cl2 (25 mL). The filtrate was washed with satd aq
NaHCO3, and water in succession before being dried
(Na2SO4) and concentrated under reduced pressure.
The crude product was purified over SiO2 (hexane–
EtOAc, 2:1) to give 5 (1.1 g) as a yellow oil in 85% yield.
25
1.2. Preparation of HClO4–SiO2 catalyst
½aꢁD ꢀ4.2 (c 1.0, CHCl3); IR (Neat): 2362, 1752, 1595,
1380, 1228, 1044, 559 cmꢀ1
;
1H NMR (300 MHz,
HClO4 (1.8 g, 12.5 mmol, as a 70% aq solution) was
added to a suspension of SiO2 (230–400 mesh, 23.7 g)
in Et2O (70.0 mL). The mixture was concentrated and
the residue was heated at 100 °C for 72 h under vacuum
to furnish HClO4–SiO2 (0.5 mmol/g) as a free flowing
powder. Caution! Although no explosions were reported
under these conditions, special care should be taken for
large-scale preparation of this reagent.
CDCl3): d 5.36 (d, J = 3.3 Hz, 1H, H-4), 5.23 (dd,
J = 8.1 and 2.4 Hz, 1H, H-20), 5.21–5.18 (m, 1H, H-
40), 5.14 (dd, J = 7.8 and 10.5 Hz, 1H, H-2), 4.99 (dd,
J = 10.3 and 3.3 Hz, 1H, H-3), 4.98–4.92 (m, 1H, H-
30), 4.62 (d, J = 7.8 Hz, 1H, H-10), 4.37 (d, J = 8.1 Hz,
1H, H-1), 3.85 (m, 2H, H-6ab), 3.80–3.72 (m, 2H, H-5,
H-50), 3.76 (s, 3H, COOCH3), 3.52 (s, 3H, OCH3),
2.14, 2.05, 2.03, 2.02, 2.01, 1.96 (6s, 18H, 6COCH3);
13C NMR (75 MHz, CDCl3): d 170.3 (3C), 169.6 (2C),
169.5 (2C), 102.3 (C-1), 100.6 (C-10), 72.7, 72.4, 72.2,
71.2 (2C), 69.5, 69.1, 67.8, 67.7, 57.2, 53.1, 21.0 (2C),
20.8 (2C), 20.7 (2C); ESIMS: m/z 659.2 [M+Na]+. Anal.
Calcd for C26H36O18: C, 49.06; H, 5.70. Found: C,
48.83; H, 5.95.
1.3. Methyl 2,3,4-tri-O-acetyl-b-D-galactopyranoside (2)
To a solution of 1 (5.0 g, 25.7 mmol) in pyridine
(50.0 mL) was added trityl chloride (10.0 g, 35.8 mmol)
and the reaction mixture was stirred at rt for 48 h in
the dark. After cooling the reaction mixture to 0 °C, ace-
tic anhydride (40.0 mL) was added and the solution was
stirred at rt for 12 h. The solvents were removed under
reduced pressure and the crude reaction mixture was
re-dissolved in CH3CN (50.0 mL). To this solution was
added HClO4–SiO2 (2.0 g, 1.0 mmol) and the mixture
was stirred at rt for 30 min. After completion of the
reaction by TLC, the mixture was filtered through Celite
and the filtrate was concentrated to dryness. Column
chromatography of the crude product over SiO2 (hex-
ane–EtOAc, 1:1) furnished 2 (6.9 g) as a yellow oil in
1.5. Methyl (b-D-glucopyranosyluronic acid)-(1!6)-b-D-
galactopyranoside (6)
A solution of 5 (500 mg, 0.78 mmol) in 0.05 M NaOCH3
in CH3OH (5.0 mL) was stirred at rt for 6 h and then
five drops of water were added and the mixture was stir-
red for another 12 h at rt. The solution was neutralized
with Dowex 50W-X8 (H+), filtered, and the filtrate was
evaporated to dryness to give a glassy solid, which was
further dissolved in aqueous CH3OH and passed
through a short column of Dowex 50W-X8 (Na+) and
concentrated under reduced pressure. The crude product
25
85% overall yield. ½aꢁD +4.8 (c 1.0, CHCl3); IR (Neat):
1748, 1372, 1226, 1061, 750 cmꢀ1 1H NMR (300
;

G. Agnihotri, A. K. Misra / Carbohydrate Research 341 (2006) 2420–2425
2423
was further purified through Sephadex LH-20 using 80%
aqueous EtOH as eluant to give pure disaccharide 6 as
its sodium salt (275 mg) in 90% yield. Glassy solid;
this compound (4.97 g, 9.42 mmol) in 0.05 M sodium
methoxide in CH3OH (100 mL) was stirred at rt for
20 min. The reaction mixture was neutralized with Dow-
ex 50W-X8 (H+) resin, filtered and evaporated to dry-
ness. The dried mass thus obtained was re-dissolved in
anhydrous CH3CN (30 mL) and benzaldehyde dimethyl-
acetal (1.7 mL, 11.3 mmol) was added followed by
HClO4–SiO2 (300 mg). After stirring at rt for 2 h, the
reaction mixture was cooled to 0 °C and acetic anhy-
dride (5.0 mL) was added and stirring continued at
0 °C for 1 h. The solvent was removed under reduced
pressure and the crude mass was purified over SiO2 (hex-
ane–EtOAc, 2:1) to afford 10 as a pale yellow solid
25
½aꢁD ꢀ10.0 (c 1.0, H2O); IR (KBr): 2351, 1685, 1198,
787 cmꢀ1
;
1H NMR (300 MHz, D2O): d 4.82 (d,
J = 8.1 Hz, 1H, H-10), 4.44 (d, J = 8.1 Hz, 1H, H-1),
4.12–3.95 (m, 2H), 3.90–3.85 (m, 3H), 3.80 (s, 1H),
3.62–3.60 (m, 2H), 3.60 (s, 3H, OCH3), 3.95–3.16 (m,
2H); 13C NMR (75 MHz, D2O): d 173.0, 102.9 (C-1),
101.6 (C-10), 74.0, 72.3, 71.5 (2C), 70.3 (2C), 70.0,
68.5, 67.0, 57.0; ESIMS: m/z 415.2 [M+Na]+. Anal.
Calcd for C13H21NaO12: C, 39.80; H, 5.40. Found: C,
39.52; H, 5.72.
25
(4.25 g, 85%). Mp 114 °C; ½aꢁD +39.8 (c 1.0, CHCl3);
1.6. Methyl 2,3,4-tri-O-acetyl-b-D-glucopyranoside (8)
IR (KBr): 2363, 2340, 1717, 1594, 1379, 1224, 1099,
754 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.85–7.80
;
To a solution of 7 (5.0 g, 25.7 mmol) in pyridine (50 mL)
was added trityl chloride (10 g, 35.8 mmol) and the reac-
tion mixture was stirred at rt for 48 h in the dark. After
cooling the reaction mixture to 0 °C, acetic anhydride
(40 mL) was added and the reaction mixture was stirred
at rt for 12 h. The solvent was removed under reduced
pressure and the crude product was re-dissolved in
CH3CN (50 mL) before HClO4–SiO2 (2.0 g, 1.0 mmol)
was added. After stirring for 30 min, the reaction was fil-
tered through Celite and concentrated to dryness. Col-
umn chromatography of the crude product over SiO2
(hexane–EtOAc, 1:1) furnished 8 (6.5 g) as a yellow oil
(m, 2H, aromatic protons), 7.76–7.70 (m, 2H, aromatic
protons), 7.42–7.27 (m, 10H, aromatic protons), 5.89–
5.80 (t, J = 9.5 Hz, 1H, H-3), 5.80 (d, J = 9.0 Hz, 1H,
H-1), 5.50 (s, 1H, CHC6H5), 4.40 (t, J = 10.1 Hz, 1H,
H-4), 4.30 (t, J = 10.1 Hz, 1H, H-2), 3.82–3.72 (m, 3H,
H-5 and H-6ab), 1.87 (s, 3H, 3COCH3); 13C NMR
(75 MHz, CDCl3): d 170.5, 168.2, 166.6, 137.3, 134.8,
134.6, 133.5 (2C), 132.1, 131.6, 129.5 (2C), 129.4 (2C),
128.7 (2C), 128.6 (2C), 126.6 (2C), 124.1, 102.1
(CHC6H5), 84.3, 79.5, 70.9 (2C), 69.0, 54.7, 20.9;
ESIMS: m/z 554 [M+Na]+. Anal. Calcd for
C29H25NO7S: C, 65.52; H, 4.74. Found: C, 65.36; H,
4.86.
25
in 80% overall yield. ½aꢁD +123.6 (c 1.0, CHCl3); IR
(Neat): 2941, 1750, 1372, 1226, 1041, 769 cmꢀ1 1H
;
NMR (300 MHz, CDCl3): 5.54 (t, J = 9.9 Hz, 1H, H-
3), 5.03 (t, J = 9.9 Hz, 1H, H-4), 4.97 (d, J = 3.6 Hz,
1H, H-1), 4.87 (dd, J = 10.5 and 3.6 Hz, 1H, H-2),
3.81–3.70 (m, 2H, H-6ab), 3.62–3.57 (m, 1H, H-5), 3.41
(s, 3H, OCH3), 2.08, 2.06, 2.02 (3s, 9H, 3COCH3);
13C NMR (75 MHz, CDCl3): d 170.8, 170.4, 170.2,
96.9 (C-1), 71.2, 70.8, 70.0, 69.4, 61.4, 55.6, 20.9
(2C), 20.8; ESIMS: m/z 343.1 [M+Na]+. Anal. Calcd
for C13H20O9: C, 48.75; H, 6.29. Found: C, 48.64; H,
6.52.
1.8. Methyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-N-
phthalimido-b-D-glucopyranosyl-(1!6)-2,3,4-tri-O-acet-
yl-a-D-glucopyranoside (11)
To a solution of 8 (1.0 g, 3.12 mmol) and 10 (2.0 g,
3.76 mmol) in anhydrous CH2Cl2 (40 mL) was added
˚
powdered 4 A molecular sieves (3.0 g) and the mixture
was stirred under argon for 1 h. After cooling the reac-
tion mixture to 0 °C, NIS (1.27 g, 5.64 mmol) and
HClO4–SiO2 (300 mg) were added and stirring was con-
tinued for 1.5 h at 0 °C. The reaction mixture was fil-
tered through Celite. The filtrate was washed with 5%
aq Na2S2O3, satd aq NaHCO3, and water, dried
(Na2SO4), and concentrated to a syrupy product. Col-
umn chromatography of the crude product over SiO2
(hexane–EtOAc, 2:1) afforded 11 (1.85 g, 80%) as a
1.7. Phenyl 3-O-acetyl-2-deoxy-4,6-O-benzylidene-2-N-
phthalimido-1-thio-b-D-glucopyranoside (10)
A solution of 9 (5.0 g, 10.47 mmol) and thiophenol
(1.6 mL, 15.71 mmol) in CH2Cl2 (50 mL) was cooled
to 0 °C. To this solution was added BF3ÆOEt2 (3.3 mL,
26.0 mmol) and the reaction mixture was stirred at
0 °C for 5 h. The reaction mixture was diluted with
CH2Cl2 (100 mL) and washed with water, satd aq NaH-
CO3, and water in succession. The organic layer was
dried (Na2SO4) and concentrated under reduced pres-
sure to give crude thiophenyl glycoside, which was puri-
fied over SiO2 (hexane–EtOAc, 2:1) to give phenyl 3,4,6-
tri-O-acetyl-2-deoxy-2-N-phthalimido-1-thio-b-D-gluco-
pyranoside as white crystals (4.97 g, 90%). A solution of
25
glassy solid; ½aꢁD +40.2 (c 1.0, CHCl3); IR (KBr):
1752, 1718, 1629, 1594, 1368, 1224, 1080, 1037, 757,
722 cmꢀ1 1H NMR (300 MHz, CDCl3): d 7.86–7.83
;
(m, 2H, aromatic protons), 7.74–7.71 (m, 2H, aromatic
protons), 7.46–7.44 (m, 2H, aromatic protons) 7.36–
7.26 (m, 3H, aromatic protons), 5.85 (t, J = 9.9 Hz,
1H, H-30), 5.54 (s, 1H, CHC6H5), 5.48 (d, J = 8.4 Hz,
1H, H-10), 5.30 (t, J = 9.6 Hz, 1H, H-3), 4.80 (t,
J = 9.9 Hz, 1H, H-4), 4.70 (dd, J = 10.2 and 3.6 Hz,
1H, H-2), 4.53 (d, J = 3.3 Hz, 1H, H-1), 4.43 (dd,

2424
G. Agnihotri, A. K. Misra / Carbohydrate Research 341 (2006) 2420–2425
J = 9.6 and 3.6 Hz, 1H, H-40), 4.28 (t, J = 8.7 Hz, 1H,
H-20), 3.94–3.71 (m, 5H, H-6ab, H-60ab, H-50), 3.50
(dd, J = 10.5 and 6.6 Hz, 1H, H-5), 3.07 (s, 3H,
OCH3), 2.01, 1.92, 1.89, 1.85 (4s, 12H, 4 COCH3); 13C
NMR (75 MHz, CDCl3): d 170.5, 170.4 (2C), 169.7,
168.2 (2C), 137.2, 134.5 (2C), 132.0, 129.6, 128.6 (3C),
126.6 (2C), 123.8 (2C), 102.0 (CHC6H5), 99.6 (C-10),
96.7 (C-1), 79.6, 71.2, 70.3, 70.1, 69.5, 69.1, 69.0, 68.0,
66.8, 55.5, 55.3, 21.0, 20.9 (2C), 20.8; ESIMS: m/z 764
[M+Na]+. Anal. Calcd for C36H39NO16: C, 58.30; H,
5.30. Found: C, 58.10; H, 5.53.
J = 8.4 Hz, 1H, H-10), 5.32 (t, J = 9.6 Hz, 1H, H-3),
4.78–4.62 (m, 4H), 4.47 (br s, 1H, H-1), 4.26 (t,
J = 9.0 Hz, 1H, H-4), 3.94–3.71 (m, 4H), 3.49 (t,
J = 9.0 Hz, 1H), 3.34 (br s, 1H, OH), 3.02 (s, 3H,
OCH3), 2.00 (s, 3H, COCH3), 1.92 (s, 6H, 2COCH3),
1.86 (s, 3H, COCH3); 13C NMR (75 MHz, CDCl3): d
171.6, 170.4 (2C), 169.8, 168.2, 167.6, 167.5, 134.6
(2C), 133.8, 132.0, 130.3 (2C), 130.2, 128.9 (3C), 123.8
(2C), 99.3 (C-10), 96.6 (C-1), 74.8, 73.6, 71.2, 70.4,
70.1, 69.6, 69.4, 68.1, 63.8, 55.2, 54.9, 21.0 (2C), 20.8
(2C); ESIMS: m/z 780.3 [M+Na]+. Anal. Calcd for
C36H39NO17 (757): C, 57.07; H, 5.19. Found: C, 58.94;
H, 5.33.
1.9. Methyl 3-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1!6)-2,3,4-tri-O-acetyl-a-D-gluco-
pyranoside (12)
1.11. Methyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyran-
osyl-(1!4)-3-O-acetyl-6-O-benzoyl-2-deoxy-2-N-phthal-
imido-b-D-glucopyranosyl-(1!6)-2,3,4-tri-O-acetyl-a-D-
glucopyranoside (15)
To a solution of 11 (1.5 g, 2.02 mmol) in CH3CN
(20 mL) was added HClO4–SiO2 (400 mg) and the mix-
ture was stirred at rt for 30 min. The reaction mixture
was filtered and concentrated to give disaccharide diol
To a solution of 13 (500 mg, 0.66 mmol) and 14 (440 mg,
25
12 (1.25 g, 95%). Glassy solid; ½aꢁD +88.2 (c 1.0, CHCl3);
1.0 mmol) in anhydrous CH2Cl2 (10 mL) was added
IR (KBr): 1749, 1718, 1662, 1386, 1226, 1039, 765 cmꢀ1
;
powdered 4 A molecular sieves (2.0 g) and the mixture
˚
1H NMR (300 MHz, CDCl3): d 7.84–7.82 (m, 2H, aro-
matic protons), 7.73–7.71 (m, 2H, aromatic protons),
5.62 (t, J = 9.0 Hz, 1H, H-30), 5.42 (d, J = 8.4 Hz, 1H,
H-10) 5.31 (t, J = 9.9 Hz, 1H, H-3), 4.80 (t, J = 9.9 Hz,
1H, H-4), 4.68 (dd, J = 10.2 and 3.3 Hz, 1H, H-2),
4.52 (d, J = 3.3 Hz, 1H, H-1), 4.19 (dd, J = 10.5 and
8.7 Hz, 1H, H-40), 4.0–3.85 (m, 3H), 3.81–3.75 (m,
2H), 3.65–3.61 (m, 1H), 3.51 (dd, J = 10.3 and 6.3 Hz,
1H), 3.05 (s, 3H, OCH3), 1.99 (s, 3H, COCH3), 1.90
(s, 6H, 2COCH3), 1.88 (s, 3H, COCH3); 13C NMR
(75 MHz, CDCl3): d 171.5, 170.4 (2C), 170.0, 168.3
(2C), 134.5 (2C), 132.0, 123.8 (3C), 98.8 (C-10), 96.7
(C-1), 76.3, 73.9, 71.2, 70.4, 70.1, 69.5, 69.1, 68.0, 62.3,
55.3, 54.9, 21.0, 20.9 (2C), 20.8; ESIMS: m/z 676.2
[M+Na]+. Anal. Calcd for C29H35NO16: C, 53.29; H,
5.40. Found: C, 53.14; H, 5.68.
was stirred under argon at rt for 1 h. The reaction mix-
ture was cooled to 0 °C and NIS (340 mg, 1.5 mmol) and
HClO4–SiO2 (50 mg) were added. After stirring at 0 °C
for 2 h, the reaction mixture was filtered through Celite
and washed with 5% aq Na2S2O3, satd aq NaHCO3, and
water in succession, before being dried (Na2SO4) and
concentrated to give a crude syrupy mass. Column puri-
fication of the crude product over SiO2 (hexane–EtOAc,
25
2:1) afforded 15 (590 mg, 82%) as a yellow oil; ½aꢁD +56
(c 1.0, CHCl3); IR (Neat): 3021, 2366, 1216, 764,
1
670 cmꢀ1; H NMR (300 MHz, CDCl3): 8.09–8.07 (m,
2H, aromatic protons), 7.86–7.83 (m, 2H, aromatic pro-
tons), 7.76–7.70 (m, 2H, aromatic protons), 7.62–7.58
(m, 1H, aromatic protons), 7.53–7.50 (m, 2H, aromatic
protons), 5.76 (t, J = 9.0 Hz, 1H, H-30), 5.51 (m, 1H,
H-200), 5.48 (d, J = 8.4 Hz, 1H, H-10), 5.40 (d,
J = 1.8 Hz, 1H, H-400), 5.30 (t, J = 9.6 Hz, 1H, H-3),
5.28 (d, J = 10.2 Hz, 1H, H-100), 5.12 (dd, J = 11.1 and
3.6 Hz, 1H, H-2), 4.79–4.70 (m, 1H, H-4), 4.66 (dd,
J = 10.2 and 3.6 Hz, 1H, H-300), 4.54 (dd, J = 12.3 and
4.5 Hz, 1H, H-20), 4.44 (d, J = 3.3 Hz, 1H, H-1), 4.25–
4.15 (m, 3H), 4.01–3.96 (m, 3H), 3.89–3.76 (m, 3H),
3.48 (dd, J = 10.5 and 7.2 Hz, 1H), 2.98 (s, 3H,
OCH3), 2.09 (s, 3H, COCH3), 1.98 (s, 6H, 2COCH3),
1.97 (s, 6H, 2COCH3), 1.90 (s, 3H, COCH3), 1.86 (s,
3H, COCH3), 1.82 (s, 3H, COCH3); 13C NMR
(75 MHz, CDCl3): d 170.9, 170.2 (2C), 170.3 (3C),
169.8, 169.3, 168.1, 168.0, 166.0, 134.2 (2C), 133.5
(2C), 131.7 (2C), 129.7 (2C), 129.4, 128.6 (2C), 123.4,
98.4 (C-10), 96.1 (C-100 and C-1), 73.9, 73.3, 72.5, 70.7,
69.9, 69.1, 68.8, 67.6 (2C), 67.3, 67.1, 66.8, 63.6, 61.1,
55.0, 54.7, 20.6 (3C), 20.5 (3C), 20.3 (2C); ESIMS: m/z
1110.4 [M+Na]+. Anal. Calcd for C50H57NO29: C,
55.20; H, 5.28. Found: C, 54.97; H, 5.54.
1.10. Methyl 3-O-acetyl-6-O-benzoyl-2-deoxy-2-phthal-
imido-b-D-glucopyranosyl-(1!6)-2,3,4-tri-O-acetyl-a-D-
glucopyranoside (13)
To a solution of 12 (1.25 g, 1.91 mmol) in CH2Cl2
(10 mL) were added pyridine (5.0 mL) and benzoyl cya-
nide (275 lL, 2.3 mmol) and the reaction mixture was
stirred at rt for 12 h. The reaction mixture was concen-
trated and purified by column chromatography over
SiO2 (hexane–EtOAc, 2:1) to give 13 (1.3 g, 90%) as a
25
glassy solid; ½aꢁD +87.0 (c 1.0, CHCl3); IR (KBr):
3783, 2922, 2364, 1750, 1386, 1226, 1043, 759 cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 8.10–8.08 (m, 2H, aro-
matic protons), 7.86–7.83 (m, 2H, aromatic protons),
7.75–7.72 (m, 2H, aromatic protons), 7.60–7.58 (m,
1H, aromatic protons), 7.51–7.46 (m, 2H, aromatic pro-
tons), 5.68 (t, J = 10.2 Hz, 1H, H-30), 5.44 (d,

G. Agnihotri, A. K. Misra / Carbohydrate Research 341 (2006) 2420–2425
2425
1.12. Methyl b-D-galactopyranosyl-(1!4)-2-acetamido-
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25
½aꢁD +30 (c 1.0, H2O); IR (KBr): 3026, 2340, 1213 cmꢀ1
;
1H NMR (300 MHz, D2O): d 4.86 (d, J = 9.0 Hz, 1H00),
4.69 (d, J = 3.0 Hz, 1H, H-1), 4.62 (d, J = 7.8 Hz, 1H,
H-10), 4.16 (d, J = 10.5 Hz, 1H, H-20), 3.99–3.85 (m,
5H), 3.81–3.70 (m, 6H), 3.66–3.63 (m, 3H), 3.58–3.48
(m, 3H), 3.34 (s, 3H, OCH3), 1.96 (s, 3H, NHAc); 13C
NMR (75 MHz, D2O): d 173.0, 101.3 (C-10 and C-100),
99.8 (C-1), 80.4, 78.6, 76.7 (2C), 75.1, 74.8, 74.0 (2C),
73.5, 72.1, 71.8, 70.7 (2C), 61.4, 61.2, 54.4, 29.0; ESIMS:
m/z 582 [M+Na]+. Anal. Calcd for C21H37NO16: C,
45.08; H, 6.67. Found: C, 44.80; H, 6.92.
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Instrumentation facilities from SAIF, CDRI are grate-
fully acknowledged. G.A. thanks CSIR, New Delhi,
for providing a Senior Research Fellowship. The
authors acknowledge Ms. Pallavi Tiwari for her techni-
cal assistance. This project was partly funded by the
Department of Science and Technology (DST), New
Delhi (Project No. SR/FTP/CSA-10/2002), India.