Synthesis of oligosaccharides corresponding to the polysaccharides of Lactobacillus and Thermophilus strains

Article abstract of DOI:10.1055/s-2007-983836

Synthesis of a branched trisaccharide and a tetrasaccharide repeating units corresponding to the polysaccharides of Lactobacillus spp. G-77 and Thermus thermophilus Samu-SA1 as their methyl glycosides has been achieved in excellent yield. Most of the glycosyl linkages are 1,2-cis in these oligosaccharide fragments. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-983836

2
660
PAPER
Synthesis of Oligosaccharides Corresponding to the Polysaccharides of
Lactobacillus and Thermophilus Strains1
O
P
ligosaccharid
i
e
s
Cor
n
respondingt
t
othe
P
u
olysaccharide
s
of
L
K
actobacillus
a
nd
T
u
hermophlilu
m
s
Strains ar Mandal, Anup Kumar Misra*
Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001, UP, India
Fax +91(522)2223938; fax +91(522)2223405; E-mail: akmisra69@rediffmail.com
Received 14 February 2007; revised 1 May 2007
ids in the adaptation process of Thermus thermophilus,
reasonable quantities of the corresponding tri- and tet-
rasaccharides are required. Recently, we engaged in the
Abstract: Synthesis of a branched trisaccharide and a tetrasaccha-
ride repeating units corresponding to the polysaccharides of Lacto-
bacillus spp. G-77 and Thermus thermophilus Samu-SA1 as their
methyl glycosides has been achieved in excellent yield. Most of the synthesis of some oligosaccharide fragments correspond-
glycosyl linkages are 1,2-cis in these oligosaccharide fragments.
ing to lactic acid bacteria and some heat-stable bacteria
for their evaluation in several biological studies.10 We
herein report a concise synthesis of a unique trisaccharide
and a tetrasaccharide as their methyl glycosides corre-
sponding to the Lactobacillus spp. G-77 and Thermus
thermophilus Samu-SA1, respectively (Figure 2).
Key words: oligosaccharide, glycoside, glycosylation, Thermus
thermophilus, Lactobacillus ssp. G-77
A wide variety of exopolysaccharides (EPS) produced by
lactic acid bacteria (LAB) present in the body provide an
important contribution to human health by acting as pre-
biotic substrates, nutraceuticals, cholesterol lowering
6)-α-D-Glcp-(1
6)-α-D-Glcp-(1
2
2
agents, or immunomodulants. Recently it has been ob-
1
α-D-Glcp
served that exopolysaccharides produced by lactic acid
bacteria have suitable rheological properties for the dairy
A
)-α-D-Galf-(1 2)-a-D-Galp-(1 6)-β-D-GlcpNAc
3
industry. In the wine industry, the polysaccharides pro-
1
duced by lactic acid bacteria can cause the alteration
known as oiliness or ropiness by increasing the consisten-
cy of the beverage. To date a number of bacteria are
known to produce exopolysaccharides in ciders and wines
2
α-D-Glcp-(1
B
and some of the structures of their exopolysaccharides Figure 1 (A) Trisaccharide repeating unit of the exopolysaccharide
4
produced by Lactobacillus spp. G-77 and (B) tetrasaccharide repea-
ting unit of the glycolipid of Thermus thermophilus Samu-SA1
have been elucidated. The structure of a ropy strain of
Lactobacillus spp. G-77 has been characterized recently5
that contains a unique 1,2-cis-linked branched trisaccha-
ride (Figure 1).
OH
O
OH
HO
HO
Another heat stable bacteria belong to the genus Thermus,
which are able to grow at extremely elevated temperature.
Some of these species have an outer membrane in the cell
envelope and can be classified as Gram-negative bacte-
O
HO
HO
HO
OH
O
HO
O
O
O
O
HO
HO
O
OCH3
NHCOCH3
HO
HO
O
O
HO
HO
HO
OCH3
OH
HO
HO
HO
6
ria. In contrast to Gram-negative bacteria, the outer mem-
O
OH
O
OH
brane of Thermus is not composed of lipopolysaccharides
but some unique glycolipids. It is believed that the gly-
7
1
2
colipids present in the outer layer of the cell envelope
Figure 2 Synthesized trisaccharide 1 and tetrasaccharide 2 as their
have an important role in its adaptation to high tempera- methyl glycosides
8
ture. Recently, the structure of a tetrasaccharide repeat-
ing unit of the glycolipid isolated from Thermus
thermophilus Samu-SA1, a thermohalophilic bacterium
has been demonstrated in which a unique D-galactofura-
nosyl moiety is present at the nonreducing end through an
a-linkage (Figure 1).
In order to synthesize the target tri- and tetrasaccharides 1
and 2, a series of differentially protected monosaccharide
derivatives were prepared from commercially available
reducing sugars (Scheme 1). Phenyl 2,3,4,6-tetra-O-ben-
zyl-1-thio-b-D-glucopyranoside (5) was prepared from
D-glucose (3) following two recently developed method-
9
11
In order to analyze and assess the role of exopolysaccha-
rides produced by Lactobacillus spp. G-77 and glycolip- ologies from our laboratory, one-pot acetylation-
12
thioglycosidation and one-pot deacetylation-benzyla-
tion.13 Methyl 3-O-benzyl-4,6-O-benzylidene-a-D-glu-
SYNTHESIS 2007, No. 17, pp 2660–2666
x
x.
x
x
.
2
0
0
7
14
copyranoside (8) was prepared from methyl a-D-
Advanced online publication: 08.08.2007
DOI: 10.1055/s-2007-983836; Art ID: P02107SS
Georg Thieme Verlag Stuttgart · New York
glucopyranoside (6) in two steps that consisted of
©

PAPER
Oligosaccharides Corresponding to the Polysaccharides of Lactobacillus and Thermophilus Strains
2661
benzylidenation15 and selective benzylation through the lation of 5 with 8 using N-iodosuccinimide (NIS) and tri-
1
6
20
formation of a stannylidene acetal. Ethyl 3,4-di-O- methylsilyl
trifluoromethanesulfonate
(TMSOTf)
acetyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimi- furnished disaccharide 19 in 80% yield. Formation of 19
do-1-thio-b-D-glucopyranoside (11) was prepared from was confirmed by its NMR and mass spectral analysis.
ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-b- Removal of the benzylidene acetal in 19 using perchloric
D-glucopyranoside (9) in three steps involving deacetyla- acid supported on silica gel21 under a modified protocol
1
7
18
tion, selective silyl group protection, and acetylation. developed in our laboratory afforded disaccharide diol 20
Phenyl 3-O-benzyl-4,6-O-benzylidene-2-O-(4-methoxy- in 90% yield. Selective glycosylation of disaccharide diol
benzyl)-1-thio-b-D-galactopyranoside (15) was prepared 20 with thioglycoside derivative 5 using N-iodosuccin-
2
0
from phenyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-galactopy- imide/trimethylsilyl trifluoromethanesulfonate
fur-
ranoside (12) in four steps involving deacetylation, ben- nished trisaccharide derivative 21 in 73% yield. Global
zylidene acetal formation, selective benzylation through debenzylation of trisaccharide derivative 21 by hydroge-
stannylidene acetal formation, and 4-methoxybenzyla- nation using hydrogen and palladium hydroxide on car-
tion. Ethyl 2,3,5,6-tetra-O-benzyl-1-thio-a-D-galactofura- bon (Pearlman’s catalyst) gave the target trisaccharide 1
noside (18) was prepared from D-galactose diethyl in 80% yield. The presence of three anomeric protons in
1
5
22
1
6
1
dithioacetal (16) by a two-step reaction sequence involv- the H NMR [d = 5.06 (d, J = 3.6 Hz, 2 H) and 4.94 (d,
ing mercury-catalyzed thiofuranoside formation19 and J = 3.6 Hz, 1 H)] and C NMR [d = 96.7 (2 C) and 96.4]
13
per-O-benzylation using benzyl bromide in the presence spectra of trisaccharide 1 supported the fact that all
of sodium hydroxide.
monosaccharide derivatives are linked together by 1,2-cis
linkages (Scheme 2). The key features of this synthetic se-
quence are the use of similar protecting groups and a high
yield in each step.
Having prepared a number of suitably functionalized
monosaccharide intermediates, synthesis of the trisaccha-
ride 1 consisting of all 1,2-cis-linked D-glucose moieties
was attempted, which is presented in Scheme 2. Glycosy-
OH
OAc
OBn
O
O
O
HO
HO
AcO
AcO
BnO
BnO
a
b
SPh
SPh
OH
OBn
OH
OAc
3
4
5
OH
Ph
Ph
O
O
O
O
O
HO
HO
c
O
d
O
HO
BnO
HO
HO
HO
OMe
OMe
OMe
6
7
8
OTBDMS
O
OTBDMS
O
OAc
AcO
AcO
HO
HO
O
SEt
AcO
AcO
e
f
SEt
SEt
N
O
N
O
N
O
O
O
O
1
1
9
10
Ph
O
Ph
Ph
O
O
O
AcO
AcO
OAc
O
O
g
i
O
O
h
O
O
SPh
SPh
BnO
SPh
HO
BnO
SPh
OAc
HO
HO
MBnO
12
13
14
15
O
O
OH OH SEt
SEt
OH
OH
OBn
OBn
18
j
HO
k
SEt
SEt
OBn
OH OH
OH
HO
BnO
16
17
Scheme 1 Reagents and conditions: (a) Ac O, BF ·OEt , 5 min, then PhSH, 5 °C, 6 h, 85%; (b) BnBr, NaOH, TBAB, THF, r.t., 8 h, 90%; (c)
2
3
2
benzaldehyde dimethyl acetal, p-TsOH, MeCN, r.t., 12 h, 90%; (d) 1. Bu SnO, MeOH, 80 °C, 2 h, 2. BnBr, CsF, DMF, r.t., 12 h, 80%; (e) 1.
2
NaOMe, MeOH, r.t., 20 min; 2. TBDMSCl, pyridine, DMAP, 50 °C, 6 h, 90% (2 steps); (f) Ac O, pyridine, r.t., 6 h, quantitative; (g) 1. NaOMe,
2
MeOH, r.t., 3 h; 2. benzaldehyde dimethyl acetal, p-TsOH, MeCN, r.t., 12 h, 85%; (h) 1. Bu SnO, MeOH, 80 °C, 2 h, 2. BnBr, CsF, DMF, r.t.,
2
1
2 h, 85%; (i) 4-methoxybenzyl chloride, NaOH, TBAB, THF, r.t., 4 h, 90%; (j) HgCl , HgO, H O, r.t., 2 h, 76%; (k) BnBr, NaOH, TBAB,
2 2
THF, r.t., 6 h, 85%.
Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York

2
662
P. K. Mandal, A. K. Misra
PAPER
OBn
O
BnO
BnO
OH
Ph
O
O
O
BnO
O
HO
O
a
b
BnO
5, a
O
c
BnO
HO
BnO
1
5
+ 8
O
O
BnO
OMe
BnO
OMe
BnO
BnO
BnO
BnO
O OMe
BnO
BnO
BnO
O
OBn
O
OBn
O
OBn
19
20
21
Scheme 2 Reagents and conditions: (a) NIS, TMSOTf, 4 Å MS, CH Cl , –45 °C, 30–45 min (80% for 19 and 73% for 21); (b) HClO on
2
2
4
silica gel, MeCN, r.t., 30 min, 90%; (c) H , 20% Pd(OH) -C, MeOH, r.t., 24 h, 80%.
2
2
13
In another set experiments, methyl a-D-galactofuranosyl- the C NMR spectra confirmed formation of the tetrasac-
charide 2. The key features of Scheme 3 include glycosy-
(
1→2)-a-D-galactopyranosyl-(1→6)-(2-acetamido-2-de-
oxy-b-D-glucopyranosyl)-(1→2)-a-D-glucopyranoside (2) lation and removal of the 4-methoxybenzyl group in one
was synthesized following Scheme 3. Condensation of 8 pot without the addition of any extra reagent and high
with 11 in the presence of N-iodosuccinimide/trimethyls- yields in the glycosylations.
ilyl trifluoromethanesulfonate furnished disaccharide 22
In summary, the synthesis of a branched trisaccharide cor-
responding to the repeating unit of the exopolysaccharide
produced by Lactobacillus spp. G-77 and a tetrasaccha-
ride corresponding to the cell-wall glycolipid of heat sta-
ble Thermus thermophilus Samu-SA1 as their methyl
glycosides has been achieved in high yield. Most of the
in 78% yield. Removal of tert-butyldimethylsilyl group23
using tetrabutylammonium fluoride in tetrahydrofuran–
acetic acid afforded disaccharide acceptor 23 in 75%
yield. Sequential glycosylation of 23 with thioglycoside
donor 15 and removal of 4-methoxybenzyl group was
2
4
achieved in one pot in the presence of N-iodosuccin-
imide/trimethylsilyl trifluoromethanesulfonate to furnish
trisaccharide acceptor 24 in 74% yield. Final glycosyla-
tion of trisaccharide derivative 24 with thioglycoside do-
nor 18 in the presence N-iodosuccinimide/trimethylsilyl
trifluoromethanesulfonate afforded the tetrasaccharide
derivative 25 in 70% yield. Removal of the N-phthalimido
monosaccharide moieties are linked together through 1,2-
cis-glycosyl linkages, glycosylation and removal of the 4-
methoxybenzyl group was achieved in one pot without the
addition of extra reagent, yields were excellent, and a gen-
eral glycosylation reaction condition has been applied.
2
5
All the reactions were monitored by TLC over silica gel coated TLC
plates; TLC was visualized by warming with 2% Ce(SO ) in 1 M
by treatment with hydrazine hydrate and N-acetylation
followed by hydrogenolysis over palladium hydroxide on
4
2
H SO sprayed plates on a hot plate. Silica gel 230–400 mesh was
2
2
2
4
carbon afforded pure tetrasaccharide 2 as its methyl gly-
1
13
used for column chromatography. H and C NMR were recorded
on Brucker Advance DPX 200 and 300 MHz using CDCl and D O
coside in 76% yield. The presence of signals for four ano-
3
2
meric protons [d = 5.81 (br s, 1 H), 5.29 (br s, 1 H), 5.10 as solvents and TMS as internal reference and acetone as external
1
(
br s, 1 H), 4.82 (d, J = 8.4 Hz, 1 H)] in the H NMR and reference. ESI-MS were recorded on a MICROMASS QUTTRO II
triple quadrupole mass spectrometer. Elementary analysis was car-
four anomeric carbons [d = 109.5, 102.7, 99.4 and 98.2] in
Ph
Ph
O
O
O
O
O
O
BnO
BnO
a
b
15,
c
8
+ 11
O
O
O
OMe
O
OMe
O
TBDMSO
AcO
O
HO
AcO
N
N
AcO
AcO
O
O
2
2
23
Ph
O
O
Ph
O
O
Ph
O
BnO
O
O
Ph
O
O
BnO
O
O
OMe
O
O
O
18,
a
d, e
O
O
OMe
O
O
O
2
N
OH O
AcO
O
BnO
BnO
AcO
BnO
OBn
BnO
N
AcO
O
AcO
O
2
4
25
O
BnO
Scheme 3 Reagents and conditions: (a) NIS, TMSOTf, 4 Å MS, CH Cl , –45 °C, 30 min (78% for 22 and 70% for 25); (b) TBAF, THF,
2
2
AcOH, r.t., 6 h, 75%; (c) NIS, TMSOTf, 4 Å MS, CH Cl , –45 °C, 30 min then 0 °C, 30 min, 74%; (d) NH NH ·H O, EtOH, 80 °C, 5 h; (ii)
2
2
2
2
2
Ac O, pyridine, r.t., 1 h; (iii) NaOMe, MeOH, r.t., 5 h, 80%; (e) H , 20% Pd(OH) -C, MeOH, r.t., 24 h, 76%.
2
2
2
Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York

PAPER
Oligosaccharides Corresponding to the Polysaccharides of Lactobacillus and Thermophilus Strains
2663
ried out on Carlo ERBA-1108 analyzer. Optical rotations were mea-
sured at 25 °C on a Rudolf Autopol III polarimeter. Commercially
available grades of organic solvents of adequate purity were used in
many reactions.
Ethyl 2,3,5,6-Tetra-O-benzyl-1-thio-b-D-galactofuranoside (18)
To a soln of D-galactose diethyl dithioacetal (16, 5.0 g, 17.5 mmol)
in H O (100 mL) was added a soln of HgCl (5.7 g, 21 mmol) in
2
2
H O (70 mL). A white precipitate was formed instantaneously. To
2
the resulting mixture 1 M NaOH was added till the pH of the soln
reached ~6. The mixture was allowed to stir at r.t. for 2 h and it was
Ethyl 3,4-Di-O-acetyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-
phthalimido-1-thio-b-D-glucopyranoside (11)
then filtered. The filtrate was made alkaline by addition of NH soln
3
A soln of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-b-
D-glucopyranoside (9, 4.0 g, 8.34 mmol) in 0.05 M NaOMe in
MeOH (50 mL) was allowed to stir at r.t. for 20 min and neutralized
and the residue was concentrated to a dry mass and the mixture was
extracted with CH Cl (250 mL). The organic layer was washed
2
2
with H O, dried (Na SO ), and concentrated under reduced pressure
2
2
4
+
with Amberlite IR-120 (H ) resin. The mixture was filtered and
to give crude ethyl 1-thio-b-D-galactofuranoside (17). To a soln of
crude 17 in THF (25 mL) were added BnBr (12.5 mL, 105 mmol),
powdered NaOH (5.6 g, 140 mmol), and TBAB (100 mg) and the
mixture was allowed to stir at r.t. for 6 h. The excess reagents were
quenched by addition of MeOH (2 mL) and the resulting soln was
concentrated under reduced pressure. The crude mass was dissolved
in CH Cl (100 mL) and the organic layer was washed with H O,
evaporated to dryness. To a soln of the crude mass in pyridine (30
mL) were added TBDMSCl (1.4 g, 9.4 mmol) and DMAP (100 mg)
and the mixture was allowed to stir at 50 °C for 6 h. After complete
consumption of the starting material, Ac O (20 mL) was added to
2
the mixture and it was allowed to stir at r.t. for 6 h. The solvents
were removed under reduced pressure to give the crude product,
which was purified by chromatography (silica gel, hexane–EtOAc,
2
2
2
dried (Na SO ), and concentrated under reduced pressure. Purifica-
2
4
5
:1) to furnish pure 11 (4.14 g, 90%) as a syrup.
tion of the crude product by chromatography (silica gel, hexane–
2
5
EtOAc, 12:1) furnished pure 18 (8.7 g, 85%) as a syrup.
[
a]D +41.5 (c 1.5, CHCl3).
2
5
–
1
[a]
D
+37.2 (c 1.5, CHCl ).
3
IR (neat): 2367, 1594, 1383, 1351, 769 cm .
1
IR (neat): 2922, 2867, 1743, 1707, 1495, 1454, 1362, 1208, 1099,
H NMR (300 MHz, CDCl ): d = 7.78–7.65 (m, 4 H, ArH), 5.76 (t,
3
–1
7
39, 698 cm .
J = 10.5, 10.5 Hz, 1 H, H3), 5.35 (d, J = 10.5 Hz, 1 H, H1), 5.06 (t,
J = 10.0, 10.0 Hz, 1 H, H4), 4.26 (t, J = 10.5, 10.5 Hz, 1 H, H2),
3
1
0
1
H NMR (300 MHz, CDCl ): d = 7.40–7.20 (m, 20 H, Ar-H), 5.38
3
.72–3.64 (m, 3 H, H5, H6 ), 2.75–2.45 (m, 2 H, SCH CH ), 1.95,
(d, J = 4.7 Hz, 1 H, H1), 4.82–4.71 (AB , J = 11.7 Hz, 2 H, PhCH ),
ab
2
3
q
2
.79 (2 s, 6 H, 2 COCH ), 1.15 (t, J = 7.4, 7.4 Hz, 3 H, SCH CH ),
4.62 (d, J = 11.7 Hz, 1 H, PhCH ), 4.54–4.46 (m, 4 H, 2 PhCH ),
3
2
3
2
2
.84 [s, 9 H, C(CH ) ], 0.02, 0.01 [2 s, 6 H, Si(CH ) ].
4.42 (d, J = 11.7 Hz, 1 H, PhCH ), 4.19–4.13 (m, 2 H, H3, H4), 4.06
3
3
3 2
2
1
3
(dd, J = 6.6, 4.7 Hz, 1 H, H2), 3.93–3.88 (m, 1 H, H5), 3.65 (dd,
J = 10.5, 4.1 Hz, 1 H, H6 ), 3.57 (dd, J = 10.5, 5.9 Hz, 1 H, H6 ),
C NMR (75 MHz, CDCl ): d = 170.3, 169.4 (2 COCH ), 167.9,
3
3
a
b
1
6
(
(
67.4 (2 CO, Phth), 134.5–124.0 (Ar-C), 80.8 (C1), 79.3, 72.2,
9.8, 63.0 (C6), 54.1 (OCH ), 26.3 [3 C, C(CH ) ], 23.9
SCH CH ), 21.0, 20.8 (2 COCH3), 18.7 [C(CH ) ], 15.3
2
.74 (dd, J = 14.7, 7.3 Hz, 2 H, SCH CH ), 1.33 (t, J = 7.4 Hz, 3 H,
2 3
3
3 3
SCH CH ).
2
3
2
3
3
3
1
3
SCH CH ), –4.9 [2 C, Si(CH ) ].
C NMR (75 MHz, CDCl ): d = 139.3–127.3 (Ar-C), 87.0 (C1),
3
2
3
3
2
ESI-MS: m/z = 574.2 [M + Na]+.
84.7, 84.0, 82.4, 78.8, 76.1, 73.6, 72.6, 72.1 (4 PhCH ), 71.1 (C6),
2
2
5.3 (SCH CH ), 15.6 (SCH CH ).
2
3
2
3
Anal. Calcd for C H NO SSi (551.2): C, 56.60; H, 6.76. Found: C,
5
2
6
37
8
+
ESI-MS: m/z = 602.2 [M + NH ] .
4
6.42; H, 6.95.
Anal. Calcd for C H O S (584.2): C, 73.94; H, 6.89. Found: C,
3
6
40
5
Phenyl 3-O-Benzyl-4,6-O-benzylidene-2-O-(4-methoxybenzyl)-
73.72; H, 7.15.
1
-thio-b-D-galactopyranoside (15)
To a soln of 14 (3.0 g, 6.6 mmol) in anhyd THF (25 mL) were added
powdered NaOH (800 mg, 20 mmol), 4-methoxybenzyl chloride
Methyl (2,3,4,6-Tetra-O-benzyl-b-D-glucopyranosyl)-(1→2)-3-
O-benzyl-4,6-O-benzylidene-a-D-glucopyranoside (19)
(1.4 mL, 9.9 mmol), and TBAB (100 mg) and the mixture was al-
To a soln of 5 (5.1 g, 8.0 mmol) and 8 (2.5 g, 6.7 mmol) in CH Cl
2
2
lowed to stir at r.t. for 4 h. The mixture was diluted with H O and
(20 mL) was added 4 Å MS (2 g) and the mixture was stirred under
argon at r.t. for 30 min. NIS (2.2 g, 9.6 mmol) was added to the mix-
ture and it was cooled to –45 °C. To the cooled mixture TMSOTf
(50 mL, 0.27 mmol) was added by injection and stirring was contin-
ued at –45 °C for 45 min. The mixture was filtered through a Celite
bed. The filtrate was washed with 5% aq Na S O , sat. NaHCO ,
2
extracted with CH Cl (3 × 50 mL). The organic layer was washed
2
2
with H O, dried (Na SO ), and concentrated under reduced pressure
2
2
4
to give the crude product, which was purified by chromatography
silica gel, hexane–EtOAc, 7:1) to afford pure 15 (3.42 g, 90%) as
a syrup.
(
2
2
3
3
2
5
and H O, dried (Na SO ), and concentrated to a syrup. Column
chromatography of the crude product by chromatography (silica
gel, hexane–EtOAc, 5:1) furnished pure 19 (4.8 g, 80%) as a syrup.
2 2 4
[
a]D –28.4 (c 1.5, CHCl3).
–
1
IR (neat): 2922, 1592, 1453, 1351, 1252, 1165, 1054, 734, 695 cm .
1
H NMR (300 MHz, CDCl ): d = 7.73–6.86 (m, 19 H, Ar H), 5.49
s, 1 H, PhCH), 4.74 (Abq, J = 12.3 Hz, 2 H, 4 MeOPhCH ), 4.65
br s, 2 H, PhCH ), 4.59 (d, J = 9.5 Hz, 1 H, H1), 4.38 (dd, J = 12.5,
.4 Hz, 1 H, H6 ), 4.14 (d, J = 3.1 Hz, 1 H, H4), 3.99 (dd, J = 12.2,
25
3
[a]D +37.6 (c 1.5, CHCl3).
(
(
1
1
2
IR (neat): 1600, 1595, 1382, 1352, 1059, 696 cm–1.
2
1
H NMR (300 MHz, CDCl ): d = 7.55–7.03 (m, 30 H, Ar-H), 5.57
a
3
.4 Hz, 1 H, H6 ), 3.90 (t, J = 9.4, 9.4 Hz, 1 H, H2), 3.83 (s, 3 H,
(s, 1 H, PhCH), 5.05 (d, J = 11.0 Hz, 1 H, PhCH ), 4.97 (d, J = 3.6
b
2
OCH ), 3.60 (dd, J = 9.2, 3.4 Hz, 1 H, H3), 3.41 (m, 1 H, H5).
Hz, 1 H, H1), 4.91–4.85 (m, 2 H, PhCH ), 4.83–4.81 (m, 2 H,
3
2
1
3
PhCH
2
), 4.80 (d, J = 1.6 Hz, 1 H, H1¢), 4.76–4.70 (m, 2 H, PhCH
2
),
C NMR (75 MHz, CDCl ): d = 159.2, 138.2–126.6 (Ar-C), 113.7,
3
4
.59–4.48 (m, 2 H, PhCH ), 4.34 (d, J = 12.0 Hz, 1 H, PhCH ), 4.30
2
2
1
(
01.2 (PhCH), 86.6 (C1), 81.4, 75.2, 75.1 (PhCH ), 73.8, 71.8
PhCH ), 69.8, 69.4 (C6), 55.2 (OCH ).
2
(
t, J = 8.4 Hz, 1 H, H3), 4.16 (dt, J = 9.3, 1.4 Hz, 2 H, H3¢, H4¢),
.92–3.85 (ddd, J = 9.5, 3.7 Hz, 2 H, H2, H2¢), 3.81 (d, J = 10.1 Hz,
1 H, H6 ), 3.74 (d, J = 10.4 Hz, 1 H, H6¢ ), 3.65–3.58 (m, 2 H, H6 ,
2
3
3
+
ESI-MS: m/z = 588.2 [M + NH ] .
4
a
a
b
H6¢ ), 3.53–3.49 (m, 1 H, H4), 3.48 (s, 3 H, OCH ), 3.45–3.39 (m,
Anal. Calcd for C H O S (570.2): C, 71.56; H, 6.0. Found: C,
7
b
3
34
34
6
2
H, H5, H5¢).
1.34; H, 6.24.
Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York

2
664
P. K. Mandal, A. K. Misra
PAPER
1
3
C NMR (75 MHz, CDCl ): d = 139.1–126.2 (Ar-C), 101.5
Methyl (3,4-Di-O-acetyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl)-(1→2)-3-O-benzyl-4,6-O-
benzylidene-a-D-glucopyranoside (22)
3
(
(
(
PhCH), 97.5 (C1), 94.5 (C1¢), 82.7, 82.2, 79.4, 77.7, 76.9, 75.9
PhCH ), 75.7 (PhCH ), 75.1 (PhCH ), 74.6, 73.3 (PhCH ), 73.0
PhCH ), 70.2, 69.2 (C6¢), 68.4 (C6), 62.5, 55.2 (OCH ).
2
2
2
2
To a soln of 8 (2.5 g, 6.7 mmol) and 11 (4.8 g, 8.7 mmol) in CH Cl
2
3
2
2
ESI-MS: m/z = 917.6 [M + Na]+.
(25 mL) was added 4 Å MS (3 g) and the mixture was stirred under
argon at r.t. for 30 min. NIS (2.3 g, 10.2 mmol) was added to the
mixture and it was cooled to –45 °C. To the cooled mixture
TMSOTf (40 mL, 0.22 mmol) was added by injection and stirring
continued at –45 °C for 30 min. The mixture was filtered through a
Celite bed. The filtrate was washed with 5% aq Na S O , sat.
Anal. Calcd for C H O (894.4): C, 73.81; H, 6.53. Found: C,
5
5
58 11
7
3.60; H, 6.90.
Methyl (2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)-(1→2)-3-
O-benzyl-a-D-glucopyranoside (20)
To a soln of 19 (4 g, 4.5 mmol) in MeCN (50 mL) was added per-
chloric acid on silica gel (1 g) and the mixture was stirred at r.t. for
2
2
3
NaHCO , and H O, dried (Na SO ), and concentrated to a syrupy
3
2
2
4
product. Column chromatography of the crude product (silica gel,
hexane–EtOAc, 5:1) furnished pure 22 (4.5 g, 78%) as a syrup.
3
0 min. The mixture was filtered through a Celite bed and evaporat-
2
5
[
a]D +24.2 (c 1.5, CHCl3).
ed to dryness. Column chromatography of the crude product (silica
gel, hexane–EtOAc, 2:1) afforded pure 20 (3.24 g, 90%) as a syrup.
IR (neat): 2932, 1753, 1719, 1597, 1461, 1384, 1355, 1237, 1052,
761 cm .
–
1
25
[
a]D +33.7 (c 1.5, CHCl3).
1H NMR (300 MHz, CDCl
): d = 7.76–6.89 (m, 14 H, ArH), 5.77
–
1
3
IR (neat): 2926, 2366, 1596, 1459, 1352, 1070, 743 cm .
(
dd, J = 9.0, 9.0 Hz, 1 H, H3¢), 5.62 (d, J = 8.4 Hz, 1 H, H1¢), 5.41
1
H NMR (300 MHz, CDCl ): d = 7.41–7.11 (m, 25 H, Ar-H), 5.04–
3
(s, 1 H, PhCH), 5.13 (t, J = 9.3, 9.3 Hz, 1 H, H4¢), 4.95 (d, J = 3.6
Hz, 1 H, H1), 4.45 (dd, J = 8.4, 8.4 Hz, 1 H, H2¢), 4.36 (d, J = 12.0
Hz, 1 H, PhCH ), 4.22 (dd, J = 9.6, 4.2 Hz, 1 H, H3), 4.18 (d,
4
.96 (m, 2 H, PhCH ), 4.92 (d, J = 3.0 Hz, 1 H, H1), 4.87–4.82 (m,
2
2
1
H, PhCH ), 4.77–4.68 (m, 4 H, H1¢, PhCH ), 4.59 (d, J = 12.0 Hz,
2 2
2
H, PhCH ), 4.51 (d, J = 11.6 Hz, 1 H, PHCH ), 4.40 (d, J = 12.0
2
2
J = 12.0 Hz, 1 H, PhCH ), 3.82 (t, J = 9.3, 9.3 Hz, 1 H, H4), 3.80–
2
Hz, 1 H, PhCH ), 4.10–3.97 (m, 2 H, H3¢, H4¢), 3.92 (t, J = 9.0, 9.0
2
3.72 (m, 4 H, H5, H6 , H6¢ ), 3.68–3.62 (m, 2 H, H2, H6¢ ), 3.50 (t,
ab
a
b
Hz, 1 H, H4), 3.85–3.75 (m, 2 H, H2, H2¢), 3.71–3.52 (m, 5 H, H3,
J = 9.3, 9.3 Hz, 1 H, H5¢), 3.44 (s, 3 H, OCH ), 2.04,1.87 (2 s, 6 H,
3
H6 , H6¢ ), 3.47 (s, 3 H, OCH ), 3.45–3.41 (m, 2 H, H5, H5¢).
ab
ab
3
2 COCH ), 0.94 [s, 9 H, C(CH ) ], 0.10, 0.09 [2 s, 6 H, Si(CH ) ].
3
3 3
3 2
1
3
C NMR (75 MHz, CDCl ): d = 138.9–127.6 (Ar-C), 96.8 (C1),
13
3
C NMR (75 MHz, CDCl ): d = 170.4, 169.5 (2 COCH ), 167.7,
3 3
9
(
(
4.0 (C1¢), 81.9, 80.4, 79.2, 77.6, 75.9 (PhCH ), 75.5 (PhCH ), 74.9
2 2
PhCH ), 74.5, 73.3 (PhCH ), 72.7 (PhCH ), 70.9, 70.7, 70.3, 68.3
C6¢), 62.4 (C6), 54.9 (OCH3).
167.6 (2 CO, Phth), 138.8–123.8 (Ar-C), 101.7 (PhCH), 100.5 (C1),
2 2 2
100.1 (C1¢), 82.5, 81.8, 76.1, 75.3, 74.2 (PhCH ), 71.5, 69.7, 69.4
2
(C6¢), 62.8 (C6), 62.6, 55.9 (C2¢), 55.2 (OCH ), 26.3 [3 C,
3
ESI-MS: m/z: 829.6 [M + Na]+.
C(CH ) ], 21.0, 20.8 (2 COCH ), 18.7 [C(CH ) ], –4.9 [2 C,
3 3 3 3 3
Si(CH ) ].
3
2
Anal. Calcd for C H O (806.4): C, 71.44; H, 6.75. Found: C,
4
8
54 11
ESI-MS: m/z = 884.3 [M + Na]+.
7
1.20; H, 7.0.
Anal. Calcd for C H NO Si (861.3): C, 62.70; H, 6.43. Found: C,
4
5
55
14
Methyl (2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl)-(1→6)-
6
2.51; H, 6.65.
[
2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-(1→2)]-3-O-ben-
zyl-a-D-glucopyranoside (21)
Methyl (3,4-Di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyra-
nosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-a-D-glucopyrano-
side (23)
To a soln of 5 (2.2 g, 3.4 mmol) and 20 (2.5 g, 3.1 mmol) in CH Cl
2
2
(
25 mL) was added 4 Å MS (3 g) and the mixture was stirred under
argon at r.t. for 30 min. NIS (925 mg, 4.1 mmol) was added to the
mixture and it was cooled to –45 °C. To the cooled mixture
TMSOTf (20 mL, 0.11 mmol) was added by injection and stirring
was continued at –45 °C for 30 min. The mixture was filtered
To a soln of 22 (3.0 g, 3.5 mmol) in AcOH (25 mL) was added 1 M
TBAF in THF (25 mL); the mixture was allowed to stir at r.t. for 6
h. The mixture was diluted with H O and extracted with CH Cl
2
2
2
(
3 × 50 mL). The organic layer was washed with sat. NaHCO and
2 2 4
3
through a Celite bed. The filtrate was washed with 5% aq Na S O ,
2
2
3
H O, dried (Na SO ), and concentrated to dryness to give the crude
sat. NaHCO , and H O, dried (Na SO ), and concentrated to a syr-
3
2
2
4
product. Column chromatography of the crude product (silica gel,
hexane–EtOAc, 3:1) furnished pure 23 (2.0 g, 75%) as a syrup.
upy product. Column chromatography of the crude product (silica
gel, hexane–EtOAc, 5:1) furnished pure 21 (3 g, 73%) as a syrup.
2
5
[
a]D –9 (c 1.5, CHCl3).
25
[
a]D +34.2 (c 1.5, CHCl3).
IR (neat): 2926, 2858, 1719, 1717, 1458, 1387, 1227, 1088, 1046,
–
1
IR (neat): 1595, 1351, 1066, 697 cm .
–1
9
94, 766 cm .
1
H NMR (300 MHz, CDCl ): d = 7.42–7.14 (m, 45 H, Ar-H), 5.06–
1
3
H NMR (300 MHz, CDCl ): d = 7.71–6.88 (m, 14 H, Ar-H), 5.65
3
5
.0 (m, 3 H, PhCH ), 4.98–4.95 (m, 2 H, PhCH ), 4.93–4.83 (m, 5
2
2
(d, J = 8.4 Hz, 1 H, H1¢), 5.62 (t, J = 8.4, 8.4 Hz, 1 H, H3¢), 5.44 (s,
1 H, PhCH), 4.96 (d, J = 3.6 Hz, 1 H, H1), 4.53–4.43 (m, 2 H, H2¢,
H, PhCH ), 4.82–4.74 (m, 4 H, H1, PhCH ), 4.70 (d, J = 2.0 Hz, 1
2
2
H, H1¢¢), 4.66 (d, J = 3.0 Hz, 1 H, H1¢), 4.60–4.50 (m, 5 H, PhCH2),
H4¢), 4.34 (d, J = 12.3 Hz, 1 H, PhCH ), 4.23 (dd, J = 9.9, 4.5 Hz, 1
2
4
3
.40–4.35 (t, J = 10.3 Hz, 1 H, H4), 4.14–3.98 (m, 2 H, H3¢, H4¢),
.97– 3.91 (m, 2 H, H2, H2¢), 3.88–3.81 (m, 4 H, H3, H3¢¢, H4¢¢,
H, H3), 4.21 (d, J = 12.3 Hz, 1 H, PhCH ), 4.10 (t, J = 7.2, 7.2 Hz,
2
1
H, H4), 3.89–3.81 (m, 1 H, H5), 3.79–3.65 (m, 5 H, H2, H6ab,
H2¢¢), 3.76–3.66 (m, 5 H, H6¢¢ , H6¢ H6 ), 3.64–3.53 (m, 4 H, H5,
a
ab,
ab
H6¢ ), 3.54–3.47 (m, 1 H, H5¢), 3.43 (s, 3 H, OCH ), 2.14, 1.94 (2
ab
3
1
3
H5¢, H5¢¢, H6¢¢ ), 3.50 (s, 3 H, OCH ). C NMR (75 MHz, CDCl3):
b
3
s, 6 H, 2 COCH3).
d = 138.1–127.9 (Ar-C), 97.6 (C1¢¢), 96.4 (C1), 94.0 (C1¢), 82.0,
1
3
C NMR (75 MHz, CDCl ): d = 171.7, 171.4 (2 COCH ), 167.9,
3
3
8
7
6
1.9, 80.4, 80.2, 80.0, 79.3, 77.7, 77.6, 76.0, 75.9, 75.5, 75.0, 74.9,
1
1
(
67.4 (2 CO, Phth), 138.7–123.8 (Ar-C), 101.7 (PhCH), 100.4 (C1),
00.3 (C1¢), 82.7, 82.2, 76.5, 74.5, 74.3 (PhCH ), 73.9, 69.9, 69.4
C6¢), 63.6 (C6), 62.6, 55.7 (C2¢), 54.9, 21.3, 21.0 (2 COCH3).
4.8, 74.4, 73.5, 73.4, 73.3 (9 PhCH ), 70.5, 70.2, 69.6, 68.8 (C6¢¢),
2
2
8.6 (C6), 68.2 (C6¢), 54.9 (OCH3).
ESI-MS: m/z = 1351.6 [M + Na]+.
ESI-MS: m/z = 770.3 [M + Na]+.
Anal. Calcd for C H O (1328.6): C, 74.08; H, 6.67. Found: C,
8
2
88 16
Anal. Calcd for C H NO (747.2): C, 62.64; H, 5.53. Found: C,
3
9
41
14
7
3.84; H, 6.95.
6
2.41; H, 6.85.
Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York

PAPER
Oligosaccharides Corresponding to the Polysaccharides of Lactobacillus and Thermophilus Strains
2665
1
3
Methyl (3-O-Benzyl-4,6-O-benzylidene-a-D-galactopyranosyl)-
C NMR (75 MHz, CDCl ): d = 170.0 (2 C, 2 COCH ), 167.9,
3
3
(
1→6)-(3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyra-
167.3 (2 C, 2 CO, Phth), 138.4–123.2 (Ar-C), 108.3 (C1¢¢¢), 101.3
(PhCH), 100.8 (PhCH), 100.0 (C1¢), 99.8 (C1), 99.4 (C1¢¢), 87.9,
82.4, 82.2, 82.0, 81.3, 76.7, 76.0 (2 C), 75.1, 74.7, 73.8, 73.7
(PhCH ), 73.4, 73.3 (PhCH ), 73.1 (PhCH ), 71.7 (PhCH ), 71.5,
nosyl)-(1→2)-3-O-benzyl-4,6-O-benzylidene-a-D-glucopyrano-
side (24)
To a soln of 23 (2.0 g, 2.7 mmol) and 15 (2.3 g, 4.0 mmol) in CH Cl
2
2
2
2
2
2
(
20 mL) was added 4 Å MS (3 g) and the mixture was stirred under
71.4 (2 C, PhCH ), 70.4 (C6¢¢¢), 69.2 (C6¢), 69.0 (2 C, C6, C6¢¢),
2
argon at r.t. for 30 min. NIS (1.1 g, 4.8 mmol) was added to the mix-
ture and it was cooled to –45 °C. To the cooled mixture TMSOTf
(
ued at –45 °C for 30 min. After consumption of 23 (monitored by
TLC) the mixture was allowed to stir at 0 °C for 20 min. The mix-
ture was filtered through a Celite bed and washed with CH Cl (50
63.7, 62.1, 55.2 (OCH ), 55.0 (C2¢¢), 20.8, 20.7 (2 COCH ).
3
3
+
ESI-MS: m/z = 1628.4 [M + NH ] .
4
40 mL, 0.22 mmol) was added by injection and stirring was contin-
Anal. Calcd for C H NO (1609.6): C, 69.35; H, 5.94. Found: C,
93 95
24
6
9.12; H, 6.20.
2
2
Methyl a-D-Glucopyranosyl-(1→6)-[a-D-glucopyranosyl-
1→2)]-a-D-glucopyranoside (1)
To a soln of 21 (1.4 g, 1 mmol) in MeOH (30 mL), was added 20%
Pd(OH) -C (500 mg) and the mixture was stirred under H at r.t. for
mL). The organic layer was washed with 5% aq Na S O , sat.
2
2
3
(
NaHCO , and H O, dried (Na SO ), and concentrated to a syrupy
3
2
2
4
product. Column chromatography of the crude product (silica gel,
hexane–EtOAc, 5:1) furnished pure 24 (2.2 g, 74%) as a syrup.
2
2
2
4 h. The mixture was filtered through Celite bed and concentrated
25
[
a]D +66.5 (c 1.5, CHCl3).
to give a solid mass that was purified through Sephadex LH-20
(80% aq EtOH) to give pure trisaccharide 1 (435 mg, 80%) as an
amorphous powder.
–
1
IR (neat): 2929, 1600, 1593, 1356, 1352, 1055, 665 cm .
1
H NMR (300 MHz, CDCl ): d = 7.67–6.86 (m, 24 H, Ar-H), 5.72
3
2
5
(
t, J = 8.8, 8.8 Hz, 1 H, H3¢), 5.66 (d, J = 8.4 Hz, 1 H, H1¢), 5.40,
[a]D +16 (c 1.0, H O).
2
5
4
4
.38 (2 s, 2 H, 2 PhCH), 5.22 (br s, 1 H, H1¢¢), 4.89 (br s, 1 H, H1),
IR (KBr): 1595, 1460, 1351, 1066, 697 cm–1.
.64–4.58 (m, 3 H, PhCH , H3¢¢), 4.38 (t, J = 9.1, 9.1 Hz, 1 H, H2¢),
2
1
H NMR (300 MHz, D O): d = 5.06 (d, J = 3.6 Hz, 2 H, H1, H1¢),
.29 (d, J = 12.5 Hz, 1 H, PhCH ), 4.22–4.02 (m, 6 H, H2¢¢, H4, H4¢,
2
2
4
3
.94 (d, J = 3.6 Hz, 1 H, H1¢¢), 4.0 (dd, J = 10.0, 3.6 Hz, 1 H, H2),
.93–3.85 (m, 2 H, H6¢¢ , H3¢¢), 3.81–3.64 (m, 7 H, H3, H4, H3¢,
H4¢¢, H6 , PhCH ), 3.98 (d, J = 12.7 Hz, 1 H, H6 ), 3.90–3.55 (m, 8
a
2
b
H, H2, H3, H5, H5¢¢, H6¢ , H6¢¢ ), 3.49–3.43 (m, 1 H, H5¢), 3.32 (s,
a
ab
ab
H4¢, H4¢¢, H5¢¢, H6¢¢ ), 3.59–3.51 (m, 4 H, H6 , H6¢ ), 3.49–3.44
(
H5¢).
3
H, OCH ), 2.13, 1.86 (2 s, 6 H, 2 COCH ).
b
ab
ab
3
3
m, 2 H, H2¢, H2¢¢), 3.43 (s, 3 H, OCH ), 3.40–3.25 (m, 2 H, H5,
1
3
3
C NMR (75 MHz, CDCl ): d = 170.4, 170.3 (2 COCH ), 167.9,
3
3
1
67.2 (2 CO, Phth), 138.3–123.4 (Ar-C), 102.2 (PhCH), 101.2
1
3
C NMR (75 MHz, D O): d = 96.7 (2 C, C1, C1¢¢), 96.4 (C1¢), 75.9,
(
PhCH), 100.6 (C1¢), 100.0 (C1¢¢), 99.5 C1), 82.1, 81.9, 77.4, 76.2,
2
7
5.5, 73.1, 72.7, 71.8 (2 C), 71.5, 71.3 (2 C), 69.9, 69.4 (2 C), 60.8
7
6
2
6.0, 73.9 (PhCH ), 73.1, 72.9 (2 C), 71.2 (PhCH ), 69.3 (C6¢¢),
2
2
(
C6¢), 60.5 (C6), 60.4 (C6¢¢), 55.1 (OCH3).
8.9 (C6¢), 67.3, 63.8, 63.1 (C6), 62.2, 55.3 (OCH ), 54.9 (C2¢),
3
+
0.8, 20.6 (2 COCH ). ESI-MS: m/z = 1110.3 [M + Na] .
ESI-MS: m/z = 541.2 [M + Na]+.
3
Anal. Calcd for C H NO (1087.4): C, 65.12; H, 5.65. Found: C,
59
61
19
Anal. Calcd for C H O (518.2): C, 44.02; H, 6.61. Found: C,
1
9
34 16
6
4.92; H, 5.90.
4
3.78; H, 6.95.
Methyl (2,3,5,6-Tetra-O-benzyl-a-D-galactofuranosyl)-(1→2)-
Methyl a-D-Galactofuranosyl-(1→2)-a-D-galactopyranosyl-
(
(
(
3-O-benzyl-4,6-O-benzylidene-a-D-galactopyranosyl)-(1→6)-
3,4-di-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-
1→2)-3-O-benzyl-4,6-O-benzylidene-a-D-glucopyranoside (25)
(
1→6)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1→2)-a-D-
glucopyranoside (2)
To a soln of tetrasaccharide derivative 25 (1.6 g, 1.0 mmol) in EtOH
(30 mL) was added hydrazine hydrate (4 mL) and the mixture was
allowed to stir at 80 °C for 5 h. The solvents were removed under
reduced pressure and to the crude mass were added pyridine (5 mL)
To a soln of 24 (2.0 g, 1.8 mmol) and 18 (1.6 g, 2.8 mmol) in CH Cl
2
2
(
20 mL) was added 4 Å MS (3 g) and the mixture was stirred under
argon at r.t. for 30 min. NIS (745 mg, 3.3 mmol) was added to the
mixture and it was cooled to –45 °C. To the cooled mixture
TMSOTf (20 mL, 0.11 mmol) was added by injection and stirring
continued at –45 °C for 30 min. The mixture was filtered through a
and Ac O (5 mL) and the mixture was kept at r.t. for 1 h. The sol-
2
vents were evaporated and co-evaporated with toluene under re-
duced pressure. To a soln of the crude mass in MeOH (25 mL) solid
NaOMe was added till pH ~10 and the mixture was allowed to stir
Celite bed. The filtrate was washed with 5% aq Na S O , sat.
2
2
3
NaHCO , and H O, dried (Na SO ), and concentrated to a syrupy
3
2
2
4
at r.t. for 5 h. The mixture was neutralized with Amberlite IR-120
product. Column chromatography of the crude product (silica gel,
hexane–EtOAc, 5:1) furnished pure 25 (2.0 g, 70%) as a syrup.
+
(
H ) resin. The mixture was filtered and evaporated to dryness. To
a soln of the crude product in MeOH (20 mL) was added 20%
25
Pd(OH) -C (500 mg) and the mixture was stirred at r.t. under a pos-
[
a]D +30.6 (c 1.5, CHCl3).
2
itive pressure of H for 24 h. The mixture was filtered through a
2
IR (neat): 2926, 2861, 2370, 1743, 1718, 1458, 1384, 1224, 1094,
Celite bed and concentrated to a crude mass. Successive purifica-
tion of the crude mass by chromatography (silica gel, CHCl3–
MeOH–H O, 10:5:1) and through a Sephadex LH-20 (MeOH–H O,
–
1
7
45 cm .
1
H NMR (300 MHz, CDCl ): d = 7.70–6.82 (m, 44 H, Ar-H), 5.80
3
2
2
(
5
4
t, J = 10.2, 10.2 Hz, 1 H, H3¢), 5.52 (d, J = 8.5 Hz, 1 H, H1¢), 5.40,
4:1) furnished pure tetrasaccharide 2 (520 mg, 76%) as an amor-
phous powder.
.37 (2 s, 2 H, 2 PhCH), 5.32 (br s, 1 H, H1¢¢¢), 5.20 (br s, 1 H, H1¢¢),
.90 (d, J = 12.2 Hz, 1 H, PhCH ), 4.85 (br s, 1 H, H1), 4.78 (d,
25
2
[
a]D +43 (c 1.0, H O).
2
J = 12.2 Hz, 1 H, PhCH ), 4.68–4.61 (m, 3 H, PhCH ), 4.56–4.47
2
2
–
1
IR (KBr): 3444, 2817, 2367, 1592, 1352, 762 cm .
(
m, 2 H, PhCH ), 4.45–4.39 (m, 3 H, H2¢, PhCH ), 4.30 (d, J = 12.0
2
2
Hz, 1 H, PhCH ), 4.28–4.18 (m, 6 H, H2¢¢, H3¢¢¢, H4, H4¢, H6¢¢¢ ,
PhCH ), 4.15–4.08 (m, 4 H, H4¢¢, H4¢¢¢, H6¢¢¢ , PhCH ), 4.05–3.90
1
2
a
H NMR (300 MHz, D O): d = 5.81 (br s, 1 H, H1¢¢), 5.29 (br s, 1
2
2
b
2
H, H1¢¢¢), 5.10 (br s, 1 H, H1), 4.82 (d, J = 8.4 Hz, 1 H, H1¢), 4.24
(t, J = 9.0, 9.0 Hz, 1 H, H4¢), 4.18–4.15 (m, 2 H, H2¢¢¢, H4¢¢), 4.10–
4.0 (m, 4 H, H3¢¢, H4, H6¢), 3.95–3.70 (m, 12 H, H3, H3¢, H3¢¢¢,
H2¢¢, H4¢¢¢, H5¢¢, H6 , H6¢¢ , H6¢¢¢ ), 3.68–3.65 (m, 1 H, H2),
(
(
(
m, 4 H, H2¢¢¢, H3¢¢, H6¢¢ ), 3.88–3.85 (m, 1 H, H5¢¢¢), 3.80–3.72
ab
m, 3 H, H3, H5, H5¢), 3.70–3.60 (m, 4 H, H6 , H6¢ ), 3.57–3.52
ab
ab
m, 1 H, H2), 3.44–3.39 (m, 1 H, H5¢¢), 3.27 (s, 3 H, OCH ), 2.02,
3
ab
ab
ab
1
.74 (2 s, 6 H, 2 COCH3).
Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York

2
666
P. K. Mandal, A. K. Misra
PAPER
3
.63–3.55 (m, 2 H, H5, H5¢, H5¢¢¢), 3.45 (s, 3 H, OCH ), 3.44–3.42
(6) (a) Quintela, J. C.; Pittenauer, E.; Allmaier, G.; Arán, V.; de
Pedro, M. A. J. Bacteriol. 1995, 177, 4947. (b) Quintela, J.
C.; Garcia-Del Portillo, F.; Pittenauer, E.; Allmaier, G.; de
Pedro, M. A. J. Bacteriol. 1999, 181, 334.
3
(
m, 1 H, H2), 2.08 (s, 3 H, NHCOCH ).
3
1
3
C NMR (75 MHz, D O): d = 170.3 (COCH ), 109.5 (C1¢¢¢), 102.7
2
3
(
C1¢), 99.4 (C1), 98.2 (C1¢¢), 83.7 (C3¢¢¢), 81.5 (C2), 81.2 (C4¢¢),
(
7) (a) Pask-Hughes, R. A.; Shaw, N. J. Bacteriol. 1982, 149,
4. (b) Lu, T. L.; Chen, C. S.; Yang, F. L.; Fung, J. M.; Chen,
7
7.2 (C2¢¢¢), 75.4 (C4¢), 75.3 (C4¢¢¢), 74.8 (C3), 73.8 (C2¢¢), 72.0 (2
C, C3¢, C5¢¢¢), 71.9 (C5), 71.4 (C5¢¢), 70.1 (C4), 69.8 (C3¢¢), 69.0
C5¢), 63.0 (C6¢¢), 61.6 (C6), 61.3 (C6¢¢¢), 61.1 (C6¢), 57.0 (C2¢),
5.3 (OCH ), 20.8 (COCH ).
5
M. Y.; Tsay, S. S.; Li, J.; Zou, W.; Wu, S. H. Carbohydr.
Res. 2004, 339, 2593.
8) Wait, R.; Carreto, L.; Nobre, M. F.; Ferreira, A. M.; Da
Costa, M. S. J. Bacteriol. 1997, 179, 6154.
9) Leone, A.; Molinaro, S.; Lindner, B.; Romano, I.; Nicolaus,
B.; Parrilli, M.; Lanzetta, R.; Holst, O. Glycobiology 2006,
(
5
3
3
(
(
ESI-MS: m/z = 744.3 [M + Na]+.
Anal. Calcd for C H NO (721.2): C, 44.94; H, 6.56. Found: C,
4
2
7
47
21
4.70; H, 6.85.
16, 766.
(
10) (a) Agnihotri, G.; Misra, A. K. Tetrahedron Lett. 2006, 47,
Acknowledgment
8493. (b) Agnihotri, G.; Misra, A. K. Carbohydr. Res. 2006,
341, 2420. (c) Tiwari, P.; Puri, A.; Chander, R.; Bhatia, G.;
Instrumentation facilities from SAIF, CDRI is gratefully acknow-
ledged. P.K.M. thanks CSIR, New Delhi for providing a Junior Re-
search fellowship. Financial support from DST, New Delhi in the
form of Ramanna Fellowship (AKM) is acknowledged.
Misra, A. K. Bioorg. Med. Chem. Lett. 2006, 16, 6028.
11) Zhang, Z.; Magnusson, G. Carbohydr. Res. 1996, 295, 41.
12) Agnihotri, G.; Tiwari, P.; Misra, A. K. Carbohydr. Res.
(
(
2005, 340, 1393.
(13) Madhusudan, S. K.; Agnihotri, G.; Negi, D. S.; Misra, A. K.
Carbohydr. Res. 2005, 340, 1373.
14) Wu, X.; Kong, F.; Lu, D.; Zhang, P. Carbohydr. Res. 1992,
229, 75.
(15) Magnusson, G.; Ahlfors, S.; Dahmen, J.; Jansson, K.;
Nilsson, U.; Noori, G.; Stenvall, K.; Tjornebo, A. J. Org.
Chem. 1990, 55, 3932.
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Synthesis 2007, No. 17, 2660–2666 © Thieme Stuttgart · New York