Efficient synthesis of two sialylated tetrasaccharides found in goat milk

Article abstract of DOI:10.1055/s-0028-1088032

Two regioisomeric sialylated tetrasaccharides found in goat milk have been obtained in excellent yield by a concise synthesis using a common disaccharide intermediate. Regioselective glycosylations and a minimal number of protecting-group manipulations ar

Full text of DOI:10.1055/s-0028-1088032

1348
PAPER
Efficient Synthesis of Two Sialylated Tetrasaccharides Found in Goat Milk
Synthesis of
T
i
w
o
Sia
n
lylated
T
etra
t
saccha
u
K
t
Milk umar Mandal, Anup Kumar Misra*
Division of Molecular Medicine, Bose Institute, A. J. C. Bose Centenary Campus, P-1/12, C.I.T. Scheme VII-M, Kolkata 700054, India
Fax +91(33)23553886; E-mail: akmisra69@rediffmail.com
Received 24 November 2008
HO
OH
Abstract: Two regioisomeric sialylated tetrasaccharides found in
goat milk have been obtained in excellent yield by a concise synthe-
sis using a common disaccharide intermediate. Regioselective gly-
O
C
O
HO
HO
HO
OH
cosylations and
manipulations are the key features of this synthetic strategy.
a
minimal number of protecting-group
NaO2C
O
O
B
O
HO
OPMP
A
O
OH
HO
HO
AcHN
HO
O
OH
Key words: oligosaccharides, glycosylations, sialic acid, goat
milk, tetrasaccharides
D
OH
1
HO
CO2Na
OH
Mammalian milk is an excellent source of complex oli-
gosaccharide antigens and bifidus factors for breastfed
newborns. To date, more than 130 different oligosaccha-
ride structures have been isolated and characterized from
different mammalian milks.1 For a long time, the biologi-
cal significance of a large number of complex oligosac-
charides found in milk were not properly studied because
it was thought that they are not directly related to nutrition
and were treated as byproducts formed during the synthe-
sis of milk. Recently, it has been demonstrated that milk
oligosaccharides have strong potential to inhibit patho-
genic microbial adhesion to the host cell surface, which is
the initial step of bacterial infections.2 Oligosaccharides
found in human milk can inhibit the adhesion of Strepto-
coccus pneumoniae and Haemophilus influenzae to hu-
man pharyngeal or buccal epithelial cells and Escherichia
coli adhesion to uroepithelial cells.2,3 A number of glyco-
proteins or glycolipids that function as tumor-associated
antigens are found in human milk.4 In addition, milk oli-
gosaccharides may function as prebiotics, promoting the
growth of benign microorganisms, such as Bifidobacteri-
um bifidus, within the lower gastrointestinal tract, and in-
hibiting the proliferation of pathogenic organisms.5 In
spite of several investigations, the physiological role of
the milk-derived oligosaccharides is not yet completely
established. Different types of milk have been analyzed
for their oligosaccharide structures that have immunolog-
ical properties.6 Recently, it has been reported that goat
milk contains two regioisomeric sialylated tetrasaccha-
rides.7 Although these oligosaccharides can be isolated
from the natural sources, it is essential to synthesize them
chemically to provide larger quantities for their proper
bioevaluations. We report herein the concise chemical
synthesis of two sialylated tetrasaccharides found in goat
milk (Figure 1).
HO
AcHN
O
D
O
HO
HO
OH
HO
HO
OH
O
O
O
B
C
O
O
HO
A
OPMP
OH
OH
OH
2
Figure 1 Structures of two synthesized sialylated tetrasaccharides
as their 4-methoxyphenyl glycosides found in goat milk
Two regioisomeric sialylated tetrasaccharides 1 and 2
(Figure 1) were synthesized from a common disaccharide
derivative 8 in a very concise manner (Scheme 1 and
Scheme 2). For this purpose, a number of suitably protect-
ed monosaccharide synthons 3,8 4,9 5,10 6,11 and 712
(Figure 2) were prepared from naturally available
monosaccharides according to methodologies reported in
the literature.
Ph
O
OBn
O
O
HO
BnO
O
OPMP
AcO
SEt
BnO
AcO
4
3
OBn
O
OAc
BnO
AcO
AcO
O
BnO
OC(=NH)CCl3
AcO
SEt
AcO
AcO
5
6
OAc
CO2Me
SMe
AcO
AcHN
O
AcO
7
Figure 2 Suitably protected monosaccharide intermediates used for
the preparation of compounds 1 and 2
SYNTHESIS 2009, No. 8, pp 1348–1354
x
x
.
x
x
.2
0
0
9
Advanced online publication: 25.03.2009
DOI: 10.1055/s-0028-1088032; Art ID: P13208SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of Two Sialylated Tetrasaccharides Found in Goat Milk
1349
R2O
AcO
Ph
O
OR1
O
OBn
O
a
3 + 4
O
O
BnO
OPMP
OBn
a
AcO
8
OBn
O
O
HO
O
BnO
OPMP
OH
8 R1,R2 = CHPh
9 R1 = R2 = H
OBn
b
13
b
6
5
c
OBn
O
BnO
BnO
Ph
O
O
OR3
HO
O
OAc
AcO
AcO
OBn
OBn
O
O
O
O
O
R3O
O
BnO
OPMP
O
O
BnO
OPMP
OR3
OAc
OBn
OAc
OBn
10 R3 = Ac
11 R3 = H
14
d
c
7
e
OBn
O
HO
OH
O
BnO
BnO
OAc
AcO
AcO
OBn
O
O
O
O
BnO
O
OPMP
OAc
OH
HO
OAc
OBn
MeO2C
OAc
OBn
AcO
15
O
O
O
BnO
O
OPMP
O
d
AcO
7
OH
OBn
AcHN
AcO
12
AcO
AcO
CO2Me
OAc
O
f
AcHN
O
AcO
OAc
HO
AcO
AcO
OBn
1
O
O
O
O
O
BnO
OPMP
Scheme 1 Reagents and conditions: (a) NIS, TMSOTf, 4 Å MS,
CH2Cl2, –30 °C, 45 min, 92%; (b) HClO4·SiO2, MeCN–H2O (9:1),
r.t., 30 min, 95%; (c) 5, TMSOTf, 4 Å MS, CH2Cl2, –40 °C, 1 h, 82%;
(d) 0.1 M NaOMe, MeOH, r.t., 4 h, 100%; (e) 7, NIS, TfOH, 3 Å MS,
MeCN–CH2Cl2 (5:1), –30 °C, 20 h, 50%; (f) 1. H2, 20% Pd(OH)2/C,
MeOH, r.t., 24 h; 2. 0.1 M NaOMe, MeOH, r.t., 12 h, then H2O (5
drops), r.t., 6 h, 70%.
OAc
OAc
OBn
16
e
2
Scheme 2 Reagents and conditions: (a) 0.1 M NaOMe, MeOH, r.t.,
2 h, 100%; (b) 1. 6, NIS, TMSOTf, CH2Cl2, 4 Å MS, –50 °C, 45 min;
Condensation of monosaccharide acceptor 3 with thiogly-
coside donor 4 in the presence of a combination of N- 2. Ac2O, py, r.t., 2 h, 74% (2 steps); (c) HClO4·SiO2, MeCN–H2O
(9:1), r.t., 30 min, 92%; (d) 7, NIS, TfOH, 3 Å MS, MeCN–CH2Cl2
(5:1), –30 °C, 20 h, 67%; (e) 1. H2, 20% Pd(OH)2/C, MeOH, r.t., 24
h; 2. 0.1 M NaOMe, MeOH, r.t., 12 h, then H2O (5 drops), r.t., 6 h,
72%.
iodosuccinimide and trimethylsilyl trifluoromethane-
sulfonate13 furnished the disaccharide derivative 8 in 92%
yield; this was the common starting point for the synthesis
of both 1 (Scheme 1) and 2 (Scheme 2). The presence of
signals at d = 5.46 (s, PhCH), 4.86 (d, J = 7.4 Hz, H-1A),
(C-1B) supported its formation. Compound 10 was
and 4.70 (d, J = 8.1 Hz, H-1B) in the 1H NMR and at d =
deacetylated with sodium methoxide, to furnish trisaccha-
102.8 (C-1A), 101.0 (PhCH), and 100.8 (C-1B) in the
ride triol acceptor 11 in quantitative yield, to be used for
glycosylation with the sialic acid derivative. Stereo- and
regioselective glycosylation16 of sialic acid derived
thioglycoside donor 7 with compound 11 in the presence
of the N-iodosuccinimide–trifluoromethanesulfonic acid
combination13 in a mixed solvent (MeCN–CH2Cl2, 5:1)
furnished sialylated tetrasaccharide derivative 12 in 50%
yield. Hydrogenolysis of tetrasaccharide derivative 12
over Pearlman’s catalyst17 followed by conventional sa-
ponification furnished tetrasaccharide 1 as its sodium salt
and 4-methoxyphenyl glycoside in 80% overall yield; it
was purified over Sephadex LH-20 gel (MeOH–H2O, 4:1)
13C NMR spectra confirmed its formation.
Removal of the benzylidene acetal from compound 8 by
use of perchloric acid supported on silica14 at room tem-
perature resulted in the formation of disaccharide diol de-
rivative 9 in 95% yield (Scheme 1). Regioselective
glycosylation of compound 9 with D-galactose-derived
trichloroacetimidate derivative 5 in the presence of tri-
methylsilyl trifluoromethanesulfonate15 afforded expected
1
trisaccharide derivative 10 in 82% yield. Presence of H
NMR signals at d = 4.86 (d, J = 8.0 Hz, H-1A), 4.61 (d,
J = 3.9 Hz, H-1B), and 4.30 (d, J = 8.0 Hz, H-1C) and 13
C
NMR signals at d = 102.9 (C-1A), 101.3 (C-1C), and 100.2
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York

1350
P. K. Mandal, A. K. Misra
PAPER
able grades of organic solvents of adequate purity were used in
many reactions.
(Scheme 1). The presence of signals at d = 4.79 (d, J = 7.3
Hz, H-1A), 4.42 (d, J = 7.9 Hz, H-1B), 4.28 (d, J = 7.0 Hz,
H-1C), 2.74 (dd, J = 12.1, 3.7 Hz, 1 H, H-3eD), and 1.87 (t,
J = 11.8 Hz, 1 H, H-3aD) in the 1H NMR and at d = 104.3
(C-2D), 104.2 (2 C, C-1C, C-1B), and 101.9 (C-1A) in the
13C NMR spectra confirmed the formation of compound
1.
4-Methoxyphenyl (2,3-Di-O-acetyl-4,6-O-benzylidene-b-D-ga-
lactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(8)
To a soln of 3 (5 g, 9 mmol) and thioglycoside donor 4 (4.3 g, 10.8
mmol) in CH2Cl2 (50 mL) was added 4 Å MS (5 g), and the mixture
was allowed to stir at r.t. under argon for 30 min. The mixture was
cooled to –30 °C and NIS (3.2 g, 14 mmol) and TMSOTf (30 mL)
were added. After stirring of the mixture at the same temperature for
45 min, it was filtered through a Celite bed and washed with CH2Cl2
(100 mL). The organic layer was washed with 5% Na2S2O3 (100
mL), sat. NaHCO3 (100 mL), and H2O (100 mL) in succession,
dried (Na2SO4), and evaporated to dryness. The crude mass was pu-
rified by chromatography (silica gel, hexane–EtOAc, 5:1); this gave
pure 8.
For the synthesis of compound 2 (Scheme 2), disaccha-
ride derivative 8 was deacetylated with sodium methoxide
to afford disaccharide diol acceptor 13 in quantitative
yield. Compound 13 was regioselectively glycosylated
with thioglycoside 6 under N-iodosuccinimide–trimethyl-
silyl trifluoromethanesulfonate catalysis; conventional
acetylation followed, to furnish trisaccharide derivative
14 in 74% overall yield. Presence of signals at d = 4.80 (d,
J = 7.5 Hz, H-1A), 4.62 (d, J = 7.9 Hz, H-1B), and 4.58 (d,
J = 8.0 Hz, H-1C) in the 1H NMR and at d = 102.7 (C-1A),
101.6 (C-1B), and 100.9 (C-1C) in the 13C NMR spectra
confirmed its formation. Removal of the benzylidene ace-
tal from compound 14 was achieved by use of perchloric
acid supported on silica at room temperature; this gave
trisaccharide diol derivative 15 in 92% yield. Stereo- and
regioselective glycosylation of trisaccharide acceptor 15
with sialic acid thioglycoside derivative 7 in the presence
of the N-iodosuccinimide–trifluoromethanesulfonic acid
combination in a mixed solvent (MeCN–CH2Cl2, 5:1) fur-
nished 6-O-a-sialylated tetrasaccharide derivative 16 in
67% yield. Hydrogenolysis of tetrasaccharide derivative
16 over Pearlman’s catalyst followed by conventional sa-
ponification furnished tetrasaccharide 2 as its sodium salt
and 4-methoxyphenyl glycoside in 78% overall yield; it
was purified over Sephadex LH-20 gel (MeOH–H2O, 4:1)
(Scheme 2). The presence of signals at d = 4.79 (d, J = 7.3
Hz, H-1A), 4.47 (d, J = 7.3 Hz, H-1B), 4.41 (d, J = 7.5 Hz,
H-1C), 2.73 (dd, J = 12.3, 4.2 Hz, 1 H, H-3eD), and 1.75 (t,
J = 11.9 Hz, 1 H, H-3aD) in the 1H NMR and at d = 105.3
(C-1B), 103.7 (C-1C), 101.9 (C-1A), 99.5 (C-2D) in the 13C
NMR spectra supported the formation of compound 2.
Yield: 7.4 g (92%); Rf = 0.3 (hexane–EtOAc, 3:1); white solid; mp
139–41 °C; [a]D25 –4.2 (c 1.5, CHCl3).
IR (KBr): 2915, 2871, 1740, 1506, 1454, 1374, 1230, 1061, 736,
698 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.49–7.19 (m, 20 H, Ar-H), 7.02
(d, J = 9.0 Hz, 2 H, Ar-H), 6.81 (d, J = 9.0 Hz, 2 H, Ar-H), 5.46 (s,
1 H, PhCH), 5.33 (dd, J = 10.3, 8.1 Hz, 1 H, H-2B), 5.12 (d, J = 11.0
Hz, 1 H, PhCH2), 5.02 (d, J = 11.0 Hz, 1 H, PhCH2), 4.86 (d, J = 7.4
Hz, 1 H, H-1A), 4.84–4.73 (m, 4 H, H-3B, 3 PhCH2), 4.70 (d, J = 8.1
Hz, 1 H, H-1B), 4.53 (d, J = 11.9 Hz, 1 H, PhCH2), 4.25 (d, J = 3.4
Hz, 1 H, H-4B), 4.18 (d, J = 12.4 Hz, 1 H, H-6aB), 4.00 (t, J = 9.0
Hz, 1 H, H-3A), 3.87–3.75 (m, 3 H, H-6bB, H-6abA), 3.79 (s, 3 H,
OCH3), 3.72–3.66 (m, 3 H, H-2A, H-5A, H-5B), 3.52–3.46 (m, 1 H,
H-4A), 2.09, 2.00 (2 s, 6 H, 2 COCH3).
13C NMR (75 MHz, CDCl3): d = 170.6, 168.9 (2 COCH3), 155.3–
114.5 (Ar-C), 102.8 (C-1A), 101.0 (PhCH), 100.8 (C-1B), 82.9 (C-
2A), 81.7 (C-5A), 77.4 (C-3A), 75.6 (PhCH2), 75.0 (3 C, PHCH2, C-
4A, C-5B), 73.6 (PhCH2), 73.3 (C-4B), 72.2 (C-3B), 69.4 (C-2B), 68.6
(C-6B), 68.0 (C-6A), 55.6 (OCH3), 20.8 (2 C, COCH3).
ESI-MS: m/z = 913.2 [M + Na]+.
Anal. Calcd for C51H54O14 (890.35): C, 68.75; H, 6.11. Found: C,
68.58; H, 6.30.
4-Methoxyphenyl (2,3-Di-O-acetyl-b-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (9)
HClO4·SiO2 (500 mg) was added to a soln of 8 (2.5 g, 2.8 mmol) in
MeCN–H2O (9:1; 50 mL), and the mixture was allowed to stir at r.t.
for 30 min. It was then filtered through a Celite bed and evaporated
to dryness. The crude product was purified through a short pad of
silica gel (hexane–EtOAc, 1:1); this gave pure 9.
In summary, efficient syntheses of two regioisomers of
sialylated tetrasaccharides 1 and 2 found in goat milk
were achieved in excellent yield. A common disaccharide
derivative has been used as the starting point for the prep-
aration of both tetrasaccharides in a minimal number of
steps. Both tetrasaccharides contain the 4-methoxyphenyl
group as a temporary anomeric protecting group, which
can be removed by standard procedures for the prepara-
tion of glycoconjugates.
Yield: 2.2 g (95%); Rf = 0.2 (hexane–EtOAc, 1:2); white solid; mp
156–58 °C; [a]D25 –31.4 (c 1.5, CHCl3).
IR (KBr): 3432, 2870, 1743, 1508, 1455, 1373, 1229, 1059, 751,
698 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.43–7.29 (m, 15 H, Ar-H), 7.05
(d, J = 9.1 Hz, 2 H, Ar-H), 6.81 (d, J = 9.2 Hz, 2 H, Ar-H), 5.26 (dd,
J = 10.1, 8.0 Hz, 1 H, H-2B), 5.02 (d, J = 11.0 Hz, 1 H, PhCH2), 4.98
(d, J = 11.2 Hz, 1 H, PhCH2), 4.85–4.79 (m, 3 H, H-1A, PhCH2),
4.75 (d, J = 10.4, 3.6 Hz, 1 H, H-3B), 4.70 (d, J = 11.9 Hz, 1 H,
PhCH2), 4.58 (d, J = 8.0 Hz, 1 H, H-1B), 4.52 (d, J = 11.9 Hz, 1 H,
PhCH2), 4.03–3.97 (m, 2 H, H-4B, H-3A), 3.73 (s, 3 H, OCH3), 3.75–
3.65 (m, 2 H, H-6abA), 3.63–3.60 (m, 2 H, H-2A, H-5A), 3.57–3.54
(m, 2 H, H-6abB), 3.52–3.48 (m, 1 H, H-4A), 3.25–3.22 (m, 1 H, H-
5B), 2.09, 1.99 (2 s, 6 H, 2 COCH3).
All the reactions were monitored by TLC on plates coated with sil-
ica gel. The TLC spots were visualized by warming the TLC plates
sprayed with 2% Ce(SO4)2 in 2 N H2SO4 on a hot plate. Silica gel
(230–400 mesh) was used for column chromatography. 1H and 13
C
NMR, 2D COSY, and HSQC spectra were recorded on a Bruker
Avance DPX 300 MHz spectrometer; CDCl3 and D2O were used as
solvents and TMS as internal reference, unless stated otherwise.
ESI-MS was carried out on a MICROMASS QUATTRO II triple
quadrupole mass spectrometer. Elementary analysis was carried out
on a Carlo ERBA-1108 analyzer. Optical rotations were measured
at 25 °C on a Rudolf Autopol III polarimeter. Commercially avail-
13C NMR (75 MHz, CDCl3): d = 170.1, 169.3 (2 COCH3), 155.3–
114.5 (Ar-C), 102.7 (C-1A), 100.3 (C-1B), 82.5 (C-2A), 81.3 (C-5A),
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York

PAPER
Synthesis of Two Sialylated Tetrasaccharides Found in Goat Milk
1351
76.4 (C-3A), 75.3 (C-3B), 74.9 (PhCH2), 74.8 (PhCH2), 73.7 (C-4A),
73.5 (2 C, PhCH2, C-5B), 69.4 (C-2B), 68.1 (C-4B), 67.8 (C-6A), 62.1
(C-6B), 55.5 (OCH3), 20.7 (2 C, COCH3).
ESI-MS: m/z = 825.1 [M + Na]+.
(d, J = 7.7 Hz, 1 H, H-1C), 4.06–4.02 (m, 1 H, H-3A), 3.99–3.82 (m,
4 H, H-6abA, H-2C, H-6aB), 3.78–3.76 (m, 1 H, H-4B), 3.75 (s, 3 H,
OCH3), 3.68–3.66 (m, 2 H, H-2A, H-2B), 3.62–3.58 (m, 1 H, H-6bB),
3.56–3.49 (m, 3 H, H-4C, H-3B, H-6aC), 3.46–3.44 (m, 1 H, H-6bC),
3.41–3.32 (m, 4 H, H-3C, H-5C, H-4A, H-5A), 3.30–3.28 (m, 1 H, H-
5B).
13C NMR (75 MHz, CDCl3): d = 155.2–114.5 (Ar-C), 103.5 (C-1C),
102.8 (C-1A), 102.7 (C-1B), 83.0 (C-2A), 82.2 (C-5A), 81.7 (C-5C),
76.2 (C-3A), 75.5 (PhCH2), 75.0 (C-4C), 74.7 (PhCH2), 74.6
(PhCH2), 73.5 (2 C, C-4A, C-3C), 73.4 (2 C, 2PhCH2), 73.3 (C-5B),
73.1 (C-3B), 72.7 (PhCH2), 72.1 (C-2B), 71.2 (C-2C), 68.4 (C-6C),
68.3 (C-6B), 68.1 (C-4B), 67.8 (C-6A), 55.4 (OCH3).
Anal. Calcd for C44H50O14 (802.32): C, 65.82; H, 6.28. Found: C,
65.60; H, 6.50.
4-Methoxyphenyl (2-O-Acetyl-3,4,6-tri-O-benzyl-b-D-galacto-
pyranosyl)-(1→6)-(2,3-di-O-acetyl-b-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (10)
A soln of 9 (2 g, 2.5 mmol) and 5 (2 g, 3.2 mmol) in anhyd CH2Cl2
(25 mL) was cooled to –50 °C. TMSOTf (100 mL) was added, and
the mixture was stirred at –40 °C for 1 h. It was diluted with CH2Cl2
(50 mL), and the organic layer was washed with sat. aq NaHCO3
(100 mL) and H2O (100 mL) in succession, dried (Na2SO4), and
evaporated to dryness. The crude product was purified by chroma-
tography (silica gel, hexane–EtOAc, 2:1); this gave pure 10.
ESI-MS: m/z = 1168.2 [M + NH4]+.
Anal. Calcd for C67H74O17 (1150.49): C, 69.90; H, 6.48. Found: C,
69.70; H, 6.72.
4-Methoxyphenyl (Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-d-glycero-a-D-galacto-2-nonulopyranosylonate)-
(2→3)-[(3,4,6-tri-O-benzyl-b-D-galactopyranosyl)-(1→6)]-(b-D-
galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyrano-
side (12)
Yield: 2.6 g (82%); Rf = 0.4 (hexane–EtOAc, 1:2); white solid; mp
176–78 °C; [a]D25 –17.3 (c 1.5, CHCl3).
IR (KBr): 3465, 2874, 1747, 1506, 1371, 1230, 1063, 747, 698
cm–1.
To a soln of 11 (1 g, 0.87 mmol) and thioglycoside donor 7 (0.9 g,
1.7 mmol) in anhyd MeCN–CH2Cl2 (5:1; 20 mL) was added 3 Å
MS (2 g), and the mixture was allowed to stir at r.t. under argon for
30 min. The mixture was cooled to –30 °C and NIS (500 mg, 2.3
mmol) and TMSOTf (15 mL) were added. After the mixture had
stirred at the same temperature for 20 h, it was filtered through a
Celite bed and washed with CH2Cl2 (100 mL). The organic layer
was washed with 5% Na2S2O3 (100 mL), sat. aq NaHCO3 (100 mL),
and H2O (100 mL) in succession, dried (Na2SO4), and evaporated to
dryness. The crude mass was purified by chromatography (silica
gel, toluene–EtOAc, 1:2); this gave pure 12.
1H NMR (300 MHz, CDCl3): d = 7.43–7.17 (m, 30 H, Ar-H), 7.00
(d, J = 9.0 Hz, 2 H, Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 5.28 (t,
J = 9.0 Hz, 1 H, H-2C), 5.21 (t, 9.1 Hz, 1 H, H-2B), 4.97 (d, J = 11.1
Hz, 2 H, PhCH2), 4.90 (d, J = 12.1 Hz, 1 H, PhCH2), 4.86 (d, J = 8.0
Hz, 1 H, H-1A), 4.84–4.80 (m, 1 H, H-3B), 4.79 (d, J = 11.8 Hz, 1 H,
PhCH2), 4.78 (d, J = 12.0 Hz, 1 H, PhCH2), 4.71 (d, J = 12.0 Hz, 1
H, PhCH2), 4.61 (d, J = 3.9 Hz, 1 H, H-1B), 4.58–4.54 (d, J = 12.0
Hz, 3 H, PhCH2), 4.49–4.39 (m, 3 H, PhCH2), 4.30 (d, J = 8.0 Hz,
1 H, H-1C), 4.03–3.98 (m, 2 H, H-4B, H-3A), 3.85 (br s, 1 H, H-4C),
3.79 (s, 3 H, OCH3), 3.76–3.73 (m, 2 H, H-6abA), 3.71–3.59 (m, 4
H, H-6abB, H-2A, H-5C), 3.55–3.39 (m, 5 H, H-6abC, H-5A, H-3C, H-
4A), 3.32–3.26 (m, 1 H, H-5B), 2.09, 2.05, 1.99 (3 s, 9 H, 3 COCH3).
13C NMR (75 MHz, CDCl3): d = 169.8, 169.5, 169.1 (3 COCH3),
155.3–114.5 (Ar-C), 102.9 (C-1A), 101.3 (C-1C), 100.2 (C-1B), 82.8
(C-2A), 81.7 (C-5C), 80.7 (C-5A), 76.2 (C-3A), 75.7 (PhCH2), 74.9
(C-3C), 74.8 (PhCH2), 74.4 (PhCH2), 73.5 (2 C, PhCH2), 73.4 (C-
4A), 73.1 (2 C, C-3B, C-5B), 72.6 (C-4C), 72.1 (PhCH2), 71.2 (C-2C),
70.4 (C-2B), 68.3 (C-6C), 67.9 (C-6A), 66.5 (2 C, C-6B, C-4B), 55.5
(OCH3), 21.0, 20.8, 20.7 (3 C, 3 COCH3).
Yield: 650 mg (50%); Rf = 0.2 (toluene–EtOAc, 1:3); white solid;
mp 127–29 °C; [a]D25 +8.4 (c 1.5, CHCl3).
IR (KBr): 3020, 2925, 2855, 2364, 1742, 1506, 1370, 1217, 1062,
765, 669 cm–1.
1H NMR (300 MHz, CDCl3): d = 7.43–7.22 (m, 30 H, Ar-H), 7.01
(d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.1 Hz, 2 H, Ar-H), 5.46–
5.44 (m, 1 H, H-8D), 5.35–5.29 (m, 1 H, H-7D), 5.04 (d, J = 12.1 Hz,
1 H, PhCH2), 4.98 (d, J = 12.5 Hz, 1 H, PhCH2), 4.88 (d, J = 5.0 Hz,
1 H, H-4D), 4.85 (d, J = 8.6 Hz, 1 H, H-1A), 4.82 (d, J = 10.3 Hz, 1
H, PhCH2), 4.79 (d, J = 10.6 Hz, 1 H, PhCH2), 4.72–4.62 (m, 4 H,
PhCH2), 4.60 (d, J = 4.4 Hz, 1 H, H-1B), 4.54 (d, J = 11.4 Hz, 1 H,
PhCH2), 4.52 (d, J = 11.2 Hz, 1 H, PhCH2), 4.49 (d, J = 11.8 Hz, 1
H, PhCH2), 4.39 (d, J = 11.8 Hz., 1 H, PhCH2), 4.28 (dd, J = 10.1,
3.9 Hz, 1 H, H-9aD), 4.18–3.97 (m, 7 H, H-1C, H-4B, H-9bD, H-5D,
H-6D, H-3A, H-3B), 3.87–3.78 (m, 4 H, H-2C, H-2B, H-6abA), 3.75–
3.69 (m, 3 H, H-2A, H-4C, H-6aB), 3.73 (s, 3 H, OCH3), 3.66–3.64
(m, 1 H, H-6bB), 3.63 (s, 3 H, COOCH3), 3.56–3.53 (m, 2 H, H-4A,
H-6aC), 3.50–3.45 (m, 2 H, H-3C, H-6bC), 3.37–3.29 (m, 3 H, H-5A,
H-5B, H-5C), 2.71 (dd, J = 12.3, 4.5 Hz, 1 H, H-3eD), 2.10–1.89 (5
s, 15 H, 5 COCH3), 2.00–1.98 (m, 1 H, H-3aD).
13C NMR (75 MHz, CDCl3): d = 170.8, 170.4, 170.3, 169.9 (2 C),
168.1 (5 COCH3, COOCH3), 155.0–114.4 (Ar-C), 103.4 (C-1C),
102.5 (C-1A), 102.3 (C-1B), 97.5 (C-2D), 82.9 (C-2A), 82.1 (C-5A),
81.6 (C-5C), 76.8 (C-3A), 76.4 (C-4B), 76.3 (PhCH2), 75.6 (C-3C),
75.1 (C-3B), 74.8 (PhCH2), 74.6 (PhCH2), 73.4 (PhCH2), 73.2 (C-
4A), 73.1 (C-5B), 73.0 (PhCH2), 72.6 (2 C, PhCH2, C-6D), 71.2 (C-
2B), 69.9 (C-2C), 68.7 (C-6C), 68.5 (C-8D), 68.3 (C-6B), 68.2 (C-4D),
67.5 (C-6A), 66.9 (2 C, C-7D, C-4C), 62.1 (C-9D), 55.5 (OCH3), 53.0
(COOCH3), 49.3 (C-5D), 37.7 (C-3D), 23.0 (NHCOOCH3), 21.1,
20.7, 20.6, 20.5 (4 COCH3).
ESI-MS: m/z = 1299.4 [M + Na]+.
Anal. Calcd for C73H80O20 (1276.52): C, 68.64; H, 6.31. Found: C,
68.45; H, 6.55.
4-Methoxyphenyl (3,4,6-Tri-O-benzyl-b-D-galactopyranosyl)-
(1→6)-(b-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-b-D-
glucopyranoside (11)
A soln of 10 (2.5 g, 2 mmol) in 0.1 M NaOMe in MeOH (mL) was
allowed to stir at r.t. for 4 h and was then neutralized with Amber-
lite-IR 120 (H+) resin. The mixture was filtered and then evaporated
to dryness; this gave the crude product, which was passed through
a short column of silica gel (toluene–EtOAc, 1:1); this gave pure 11.
Yield: 2.3 g (100%); Rf = 0.3 (toluene–EtOAc, 1:2); white solid; mp
168–69 °C; [a]D25 +3.6 (c 1.5, CHCl3).
IR (KBr): 3449, 3020, 2926, 2364, 1505, 1216, 1064, 763, 669
cm–1.
1H NMR (300 MHz, CDCl3): d = 7.37–7.17 (m, 30 H, Ar-H), 6.98
(d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.0 Hz, 2 H, Ar-H), 4.97 (d,
J = 11.2 Hz, 1 H, PhCH2), 4.92 (d, J = 11.0 Hz, 1 H PhCH2), 4.85
(d, J = 4.86 Hz, 1 H, H-1A), 4.82–4.77 (m, 3 H, PhCH2), 4.71–4.63
(m, 3 H, PhCH2), 4.54 (d, J = 12.1 Hz, 1 H, PhCH2), 4.53 (d,
J = 11.7 Hz, 1 H, PhCH2), 4.42 (d, J = 12.4 Hz, 1 H, PhCH2), 4.41
(d, J = 6.3 Hz, 1 H, H-1B), 4.38 (d, J = 11.8 Hz, 1 H, PhCH2), 4.07
ESI-MS: m/z = 1646.5 [M + Na]+.
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York

1352
P. K. Mandal, A. K. Misra
PAPER
Anal. Calcd for C87H101NO29 (1623.64): C, 64.31; H, 6.27; found:
C, 64.12; H, 6.50.
J = 10.3, 3.5 Hz, 1 H, H-3B), 3.47–3.44 (m, 1 H, H-4A), 2.19, 2.06,
2.05, 2.04, 1.99 (5 s, 15 H, 5 COCH3).
13C NMR (75 MHz, CDCl3): d = 170.1, 170.0, 169.9, 168.9, 168.5
(5 COCH3), 155.3–114.5 (Ar-C), 102.7 (C-1A), 101.6 (C-1B), 100.9
(C-1C), 100.6 (PhCH), 82.9 (C-2A), 81.6 (C-5B), 77.3 (C-3A), 77.2
(C-3B), 75.7 (C-4B), 75.5 (PhCH2), 75.1 (C-4A), 74.9 (PhCH2), 73.6
(PhCH2), 70.9 (C-2B), 70.8 (C-3C), 70.7 (C-5C), 68.4 (2 C, C-2C, C-
5A), 67.9 (C-6B), 66.8 (C-4C), 61.1 (2 C, C-6A, C-6C), 55.4 (OCH3),
21.0, 20.6 (2 C), 20.5 (2 C) (5 COCH3).
4-Methoxyphenyl (4,6-O-Benzylidene-b-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (13)
A soln of 8 (3 g, 3.4 mmol) in 0.1 M NaOMe in MeOH (100 mL)
was allowed to stir at r.t. for 2 h and then neutralized with Amber-
lite-IR 120 (H+) resin. The mixture was filtered and evaporated to
dryness to give the crude product, which was passed through a short
column of silica gel (hexane–EtOAc, 2:1); this gave pure 13.
ESI-MS: m/z = 1201.3 [M + Na]+.
Yield: 2.73 g (100%); Rf = 0.3 (hexane–EtOAc, 1:1); yellow oil;
[a]D25 –25.3 (c 1.5, CHCl3).
Anal. Calcd for C63H70O22 (1178.43): C, 64.17; H, 5.98. Found: C,
64.0; H, 6.20.
IR (neat): 3416, 2919, 2851, 2362, 1734, 1505, 1459, 1218, 1059,
766, 698 cm–1.
4-Methoxyphenyl (2,3,4,6-Tetra-O-acetyl-b-D-galactopyrano-
syl)-(1→3)-(2-O-acetyl-b-D-galactopyranosyl)-(1→4)-2,3,6-tri-
O-benzyl-b-D-glucopyranoside (15)
HClO4·SiO2 (500 mg) was added to a soln of 14 (2 g, 1.7 mmol) in
MeCN–H2O (9:1; 50 mL), and the mixture was allowed to stir at r.t.
for 30 min. It was then filtered through a Celite bed and evaporated
to dryness. The crude product was purified through a short pad of
silica gel (toluene–EtOAc, 1:1); this gave pure 15.
1H NMR (300 MHz, CDCl3): d = 7.48–7.23 (m, 20 H, Ar-H), 7.02
(d, J = 9.0 Hz, 2 H, Ar-H), 6.82 (d, J = 9.0 Hz, 2 H, Ar-H), 5.46 (s,
1 H, PhCH), 5.10 (d, J = 11.0 Hz, 1 H, PhCH2), 5.03 (d, J = 11.0 Hz,
1 H, PhCH2), 4.95 (d, J = 11.1 Hz, 1 H, PhCH2), 4.88 (d, J = 7.3Hz,
1 H, H-1A), 4.83 (d, J = 11.0 Hz, 1 H, PhCH2), 4.71 (d, J = 12.1 Hz,
1 H, PhCH2), 4.59 (d, J = 11.0 Hz, 1 H, PhCH2), 4.56 (d, J = 7.5 Hz,
1 H, H-1B), 4.11–4.01 (m, 4 H, H-3A, H-4B, H-6aA, H-6aB), 3.89–
3.85 (m, 2 H, H-6bA, H-6bB), 3.78 (s, 3 H, OCH3), 3.76–3.72 (m, 3
H, H-2A, H-5A, H-5B), 3.69–3.56 (m, 2 H, H-2B, H-4A), 3.48–3.39
(m, 1 H, H-3B).
13C NMR (75 MHz, CDCl3): d = 155.3–114.5 (Ar-C), 103.4 (C-1B),
102.9 (C-1A), 101.2 (PhCH), 83.4 (C-2A), 81.8 (C-5A), 77.5 (C-3A),
75.5 (PhCH2), 75.2 (C-4B), 75.0 (PhCH2), 74.7 (C-2B), 73.3 (2 C,
PhCH2, C-4A), 72.8 (C-3B), 72.0 (C-5B), 68.8 (C-6A), 68.2 (C-6B),
55.4 (OCH3).
Yield: 1.7 g (92%); Rf = 0.4 (toluene–EtOAc, 1:2); colorless oil;
[a]D25 –12.3 (c 1.5, CHCl3).
IR (neat): 3482, 2920, 2361, 1749, 1506, 1370, 1225, 1062, 755
cm–1.
1H NMR (300 MHz, CDCl3): d = 7.39–7.25 (m, 15 H, Ar-H), 6.98
(d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.0 Hz, 2 H, Ar-H), 5.35–
5.34 (m, 1 H, H-4C), 5.19–5.12 (m, 2 H, H-2B, H-2C), 4.99–4.94 (m,
3 H, H-3C, 2 PhCH2), 4.81 (d, J = 8.1 Hz, 1 H, H-1A), 4.78 (d,
J = 10.6 Hz, 1 H, PhCH2), 4.76 (d, J = 12.3 Hz, 1 H, PhCH2), 4.73
(d, J = 12.0 Hz, 1 H, PhCH2), 4.53 (d, J = 8.0 Hz, 1 H, H-1B), 4.49
(d, J = 11.2 Hz, 1 H, PhCH2), 4.46 (d, J = 7.8 Hz, 1 H, H-1C), 4.19–
4.05 (m, 2 H, H-6abC), 3.94–3.89 (m, 3 H, H-4B, H-5C, H-3A), 3.77
(s, 3 H, OCH3), 3.75–3.68 (m, 2 H, H-6abA), 3.67–3.58 (m, 3 H, H-
5B, H-6aB, H-2A), 3.55–3.44 (m, 3 H, H-3B, H-6bB, H-4A), 3.32–3.28
(m, 1 H, H-5A), 2.16, 2.06, 1.98 (3 s, 15 H, 5 COCH3).
13C NMR (75 MHz, CDCl3): d = 170.2, 170.0, 169.9, 169.0, 168.6
(5 COCH3), 155.3–114.5 (Ar-C), 102.8 (C-1A), 101.5 (C-1B), 100.3
(C-1C), 82.4 (C-2A), 81.3 (C-5B), 80.1 (C-3B), 76.6 (C-3A), 75.5
(PhCH2), 75.1 (C-4A), 74.9 (PhCH2), 74.6 (C-5A), 73.6 (PhCH2),
71.1 (C-2C), 70.9 (C-3C), 70.6 (C-4B), 68.7 (C-5C), 68.5 (C-2B), 67.9
(C-6A), 66.9 (C-4C), 61.6 (C-6B), 61.3 (C-6C), 55.5 (OCH3), 20.8 (2
C), 20.6, 20.5, 20.4 (5 COCH3).
ESI-MS: m/z = 829.3 [M + Na]+.
Anal. Calcd for C47H50O12 (806.33): C, 69.96; H, 6.25. Found: C,
69.75; H, 6.50.
4-Methoxyphenyl (2,3,4,6-Tetra-O-acetyl-b-D-galactopyrano-
syl)-(1→3)-(2-O-acetyl-4,6-O-benzylidene-b-D-galactopyrano-
syl)-(1→4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside (14)
To a soln of 13 (2 g, 2.5 mmol) and thioglycoside donor 6 (1.1 g, 2.7
mmol) in CH2Cl2 (40 mL) was added 4 Å MS (5 g), and the mixture
was allowed to stir at r.t. under argon for 30 min. The mixture was
cooled to –50 °C and NIS (790 mg, 3.5 mmol) and TMSOTf (10 mL)
were added. After the mixture had stirred at the same temperature
for 30 min, it was filtered through a Celite bed and washed with
CH2Cl2 (100 mL). The organic layer was washed with 5% Na2S2O3
(100 mL), sat. aq NaHCO3 (100 mL), and H2O (100 mL) in succes-
sion, dried (Na2SO4), and evaporated to dryness. The crude mass
was acetylated with Ac2O (5 mL) and py (5 mL) at r.t. The acetylat-
ed crude mass was purified by chromatography (silica gel, hexane–
EtOAc, 3:1); this gave pure 14.
ESI-MS: m/z = 1113.2 [M + Na]+.
Anal. Calcd for C56H66O22 (1090.40): C, 61.64; H, 6.10. Found: C,
61.46; H, 6.32.
4-Methoxyphenyl (Methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonate)-
(2→6)-[(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-(1→3)]-
(2-O-acetyl-b-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-b-
D-glucopyranoside (16)
To a soln of 15 (1 g, 0.92 mmol) and thioglycoside donor 7 (960 mg,
1.8 mmol) in anhyd MeCN–CH2Cl2 (5:1; 15 mL) was added 3 Å
MS (2 g), and the mixture was allowed to stir at r.t. under argon for
30 min. The mixture was cooled to –30 °C and NIS (525 mg, 2.3
mmol) and TMSOTf (10 mL) were added. After the mixture had
stirred at the same temperature for 3 h, it was filtered through a
Celite bed and washed with CH2Cl2 (100 mL). The organic layer
was washed with 5% Na2S2O3 (100 mL), sat. aq NaHCO3 (100 mL),
and H2O (100 mL) in succession, dried (Na2SO4), and evaporated to
dryness. The crude mass was purified by chromatography (silica
gel, toluene–EtOAc, 1:2); this gave pure 16.
Yield: 2.2 g (74%); Rf = 0.4 (hexane–EtOAc, 1:1); white solid; mp
130–32 °C; [a]D25 –3.7 (c 1.5, CHCl3).
IR (KBr): 3465, 2871, 1751, 1507, 1370, 1226, 1061, 751, 699
cm–1.
1H NMR (300 MHz, CDCl3): d = 7.50–7.15 (m, 20 H, Ar-H), 6.98
(d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.0 Hz, 2 H, Ar-H), 5.50 (s,
1 H, PhCH), 5.35–5.34 (m, 1 H, H-4C), 5.29–5.23 (m, 1 H, H-2B),
5.15 (dd, J = 10.4, 8.0 Hz, 1 H, H-2C), 5.06 (d, J = 11.1 Hz, 1 H,
PhCH2), 4.98 (d, J = 11.1 Hz, 1 H, PhCH2), 4.93 (t, J = 5.3 Hz, 1 H,
H-3C), 4.82 (d, J = 11.1 Hz, 1 H, PhCH2), 4.80 (d, J = 7.5 Hz, 1 H,
H-1A), 4.78–4.71 (2 d, J = 11.8 Hz, 2 H, PhCH2), 4.62 (d, J = 7.9
Hz, 1 H, H-1B), 4.58 (d, J = 8.0 Hz, 1 H, H-1C), 4.51 (d, J = 11.9 Hz,
1 H, PhCH2), 4.24–4.08 (m, 4 H, H-6abC, H-6aA, H-4B), 3.97–3.87
(m, 3 H, H-3A, H-6bA, H-5C), 3.82–3.72 (m, 3 H, H-6abB, H-5A),
3.75 (s, 3 H, OCH3), 3.69–3.64 (m, 2 H, H-5B, H-2A), 3.60 (dd,
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York

PAPER
Synthesis of Two Sialylated Tetrasaccharides Found in Goat Milk
1353
Yield: 940 mg (67%); Rf = 0.3 (toluene–EtOAc, 1:3); white solid;
mp 142–44 °C; [a]D25 –11.4 (c 1.5, CHCl3).
4C, C-5B), 73.6 (C-5C), 73.4 (C-4D), 71.6 (2 C, C-3A, C-3C), 69.5 (C-
8D), 69.3 (C-6D), 69.0 (C-2B), 68.8 (C-6A), 68.4 (C-3B), 67.7 (C-4B),
63.7 (C-9D), 61.5 (C-6B), 61.0 (C-6C), 55.1 (OCH3), 52.7 (C-5D),
39.9 (C-3D), 22.6 (NHCOCH3).
ESI-MS: m/z = 924.1 [M + 1]+.
IR (KBr): 3486, 2925, 1748, 1507, 1371, 1226, 1064, 741, 699
cm–1.
1H NMR (300 MHz, CDCl3): d = 7.39–7.24 (m, 15 H, Ar-H), 7.00
(d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.1 Hz, 2 H, Ar-H), 5.39–
5.32 (m, 3 H, H-7D, H-8D, H-4C), 5.26–5.15 (m, 3 H, H-2C, H-2B, H-
6D), 5.02–4.93 (m, 3 H, H-3C, H-4D, PhCH2), 4.86 (d, J = 7.7 Hz, 1
H, H-1A), 4.85 (d, J = 11.3 Hz, 1 H, PhCH2), 4.77 (d, J = 11.1 Hz, 1
H, PhCH2), 4.75 (d, J = 11.0 Hz, 1 H, PhCH2), 4.69 (d, J = 8.2 Hz,
1 H, H-1B), 4.65 (d, J = 8.0 Hz, 1 H, H-5D), 4.57 (d, J = 8.0 Hz, 1 H,
H-1C), 4.53 (d, J = 12.7 Hz, 1 H, PhCH2), 4.35–4.25 (m, 1 H, H-
6aC), 4.24–4.18 (m, 1 H, H-6aB), 4.13–4.04 (m, 4 H, H-5C, H-9aD,
H-6bC, H-6bB), 3.95–3.90 (m, 3 H, H-4B, H-5A, H-9bD), 3.82–3.72
(m, 2 H, H-6abA), 3.76 (s, 6 H, OCH3 and COOCH3), 3.71–3.62 (m,
2 H, H-5B, H-2A), 3.58–3.51 (m, 2 H, H-3B, H-4A), 3.38 (t, J = 7.0
Hz, 1 H, H-3A), 2.55 (dd, J = 12.1, 4.4 Hz, 1 H, H-3eD), 2.16–1.88
(10 s, 30 H, 10 COCH3), 1.99–1.89 (m, 1 H, H-3aD).
13C NMR (75 MHz, CDCl3): d = 170.9, 170.7, 170.5, 170.3 (2 C),
170.2, 170.1, 170.0, 169.3, 168.9, 167.9 (9 COCH3, COOCH3,
NHCOCH3), 155.3–114.5 (Ar-C), 102.6 (C-1A), 101.5 (C-1C),
100.0 (C-1B), 99.0 (C-2D), 82.4 (C-2A), 81.7 (C-5B), 79.5 (C-3B),
76.4 (C-5A), 74.9 (C-4A), 74.8 (2 C, 2 PhCH2), 73.7 (C-4B), 73.6
(PhCH2), 72.9 (C-5C), 72.3 (C-3A), 71.4 (C-2B), 71.3 (C-2C), 70.8 (2
C, C-3C, C-4D), 69.3 (C-4C), 69.1 (C-6D), 68.5 (C-9D), 67.6 (C-7D),
66.8 (C-8D), 62.4 (C-6C), 62.1 (C-6B), 60.9 (C-6A), 55.6 (OCH3),
52.9 (COOCH3), 49.4 (C-5D), 37.0 (C-3D), 22.7 (NHCOCH3), 21.1,
21.0, 20.9, 20.8, 20.7, 20.6 (2 C), 20.5, 20.3 (9 COCH3).
Anal. Calcd for C36H54NNaO25 (923.28): C, 46.81; H, 5.89. Found:
C, 46.60; H, 6.15.
4-Methoxyphenyl (Sodium 5-acetamido-3,5-dideoxy-D-glycero-
a-d-galacto-2-nonulopyranosylonate)-(2→6)-[(b-D-galactopyr-
anosyl)-(1→3)]-(b-D-galactopyranosyl)-(1→4)-b-D-glucopyra-
noside (2)
To a soln of the tetrasaccharide derivative 16 (600 mg, 0.38 mmol)
in MeOH (20 mL) was added 20% Pd(OH)2/C (400 mg), and the
mixture was allowed to stir at r.t. for 24 h under a positive pressure
of H2. The mixture was filtered through a Celite bed and concentrat-
ed under reduced pressure. The crude mass was dissolved in 0.1 M
NaOMe in MeOH (30 mL), and the mixture was allowed to stir at
r.t. for 12 h; then a few drops of distilled H2O was added, and the
mixture was allowed to stir for 6 h. The mixture was neutralized
with Dowex 50W X8 (H+) resin, filtered, and evaporated to dryness,
before it was again passed through a short pad of Dowex 50W X8
(Na+) resin. The crude product was purified by being passed
through a column of Sephadex-LH-20 (MeOH–H2O, 4:1); this gave
tetrasaccharide 2 as its sodium salt.
Yield: 250 mg (72%); Rf = 0.3 (MeCN–MeOH–H2O, 4:1:0.5);
white powder; [a]D25 –8.7 (c 1.1, H2O).
IR (KBr): 3021, 2926, 2401, 1216, 767, 670 cm–1.
ESI-MS: m/z = 1581.1 [M + NH4]+.
1H NMR (300 MHz, D2O): d = 7.01 (d, J = 8.9 Hz, 2 H, Ar-H), 6.80
(d, J = 8.9 Hz, 2 H, Ar-H), 4.79 (d, J = 7.3 Hz, 1 H, H-1A), 4.47 (d,
J = 7.3 Hz, 1 H, H-1B), 4.41 (d, J = 7.5 Hz, 1 H, H-1C), 4.14–4.05
(m, 3 H, H-8D, H-7D, H-9aD), 3.91–3.76 (m, 7 H, H-4D, H-5D, H-4B,
H-6D, H-6abA, H-9bD), 3.75–3.67 (m, 3 H, H-2C, H-6abC), 3.71 (s, 3
H, OCH3), 3.64–3.57 (m, 6 H, H-3A, H-2B, H-5B, H-3C, H-6abB),
3.55–3.44 (m, 6 H, H-2A, H-4A, H-5A, H-3B, H-4C, H-5C), 2.73 (dd,
J = 12.3, 4.2 Hz, 1 H, H-3eD), 1.96 (s, 3 H, NHCOCH3), 1.75 (t,
J = 11.9 Hz, 1 H, H-3aD).
13C NMR (75 MHz, D2O): d = 174.4 (COONa), 173.9 (NHCOCH3),
155.6–114.5 (Ar-C), 105.3 (C-1B), 103.7 (C-1C), 101.9 (C-1A), 99.5
(C-2D), 83.4 (C-2C), 80.5 (C-5B), 75.7 (C-2B), 75.2 (2 C, C-3A, C-
3C), 74.1 (C-4A), 73.7 (C-5A), 73.5 (2 C, C-3B, C-4C), 71.9 (2 C, C-
4D, C-6D), 70.4 (C-4B), 69.3 (C-8D), 69.2 (C-7D), 68.8 (C-5C), 68.3
(C-2A), 63.6 (C-9D), 63.1 (C-6B), 61.5 (C-6C), 60.9 (C-6A), 55.1
(OCH3), 52.6 (C-5D), 40.8 (C-3D), 21.9 (NHCOCH3).
Anal. Calcd for C76H93NO34 (1563.56): C, 58.34; H, 5.99. Found: C,
58.13; H, 6.25.
4-Methoxyphenyl (Sodium 5-Acetamido-3,5-dideoxy-D-glyce-
ro-a-D-galacto-2-nonulopyranosylonate)-(2→3)-[(b-D-galacto-
pyranosyl)-(1→6)]-(b-D-galactopyranosyl)-(1→4)-b-D-gluco-
pyranoside (1)
To a soln of the tetrasaccharide derivative 12 (500 mg, 0.33 mmol)
in MeOH (20 mL) was added 20% Pd(OH)2/C (400 mg), and the
mixture was allowed to stir at r.t. for 24 h under a positive pressure
of H2. The mixture was filtered through a Celite bed and concentrat-
ed under reduced pressure. The crude mass was dissolved in 0.1 M
NaOMe in MeOH (30 mL), and the mixture was allowed to stir at
r.t. for 12 h; then a few drops of distilled H2O was added, and the
mixture was allowed to stir for 6 h. The mixture was neutralized
with Dowex 50W X8 (H+) resin, filtered, and evaporated to dryness
and again passed through a short pad of Dowex 50W X8 (Na+) resin.
The crude product was purified by being passed through a column
of Sephadex-LH-20 (MeOH–H2O, 4:1); this gave tetrasaccharide 1
as its sodium salt.
ESI-MS: m/z = 924.2 [M + 1]+.
Anal. Calcd for C36H54NNaO25 (923.28): C, 46.81; H, 5.89. Found:
C, 46.58; H, 6.18.
Yield: 215 mg (70%); Rf = 0.2 (MeCN–MeOH–H2O, 4:1:0.5);
white powder; [a]D25 –9.3 (c 1.1, H2O).
Acknowledgment
IR (KBr): 3443, 3021, 1637, 1216, 767, 669 cm–1.
Instrumentation facilities from SAIF, CDRI, Lucknow is gratefully
acknowledged. P.K.M. thanks CSIR, New Delhi for providing a Se-
nior Research fellowship. A.K.M. thanks the Department of Sci-
ence and Technology (DST), New Delhi for providing a Ramanna
Fellowship (SR/S1/RFPC-06/2006).
1H NMR (300 MHz, D2O): d = 7.01 (d, J = 9.0 Hz, 2 H, Ar-H), 6.80
(d, J = 9.0 Hz, 2 H, Ar-H), 4.79 (d, J = 7.3 Hz, 1 H, H-1A), 4.42 (d,
J = 7.9 Hz, 1 H, H-1B), 4.28 (d, J = 7.0 Hz, 1 H, H-1C), 4.06–3.96
(m, 2 H, H-8D, H-7D), 3.92–3.75 (m, 9 H, H-4D, H-9aD, H-6D, H-5D,
H-3A, H-6abB, H-6abA), 3.72–3.65 (m, 3 H, H-4B, H-6abC), 3.71 (s,
3 H, OCH3), 3.65–3.55 (m, 5 H, H-2B, H-9bD, H-3B, H-3C, H-4C),
3.52–3.43 (m, 6 H, H-2A, H-2C, H-5B, H-5A, H-5C, H-4A), 2.74 (dd,
J = 12.1, 3.7 Hz, 1 H, H-3eD), 1.97 (s, 3 H, NHCOCH3), 1.87 (t,
J = 11.8 Hz, 1 H, H-3aD).
References
(1) (a) Newburg, D. S.; Neubaur, S. H. Handbook of Milk
Composition; Academic Press: San Diego, 1995, 273–349.
(b) Stahl, B.; Thurl, S.; Zeng, J.; Karas, M.; Hillenkamp, F.;
Steup, M.; Sawatzki, G. Anal. Biochem. 1994, 223, 218.
13C NMR (75 MHz, D2O): d = 174.0 (COONa), 155.6–114.5 (Ar-
C), 104.3 (C-2D), 104.2 (2 C, C-1C, C-1B), 101.9 (C-1A), 81.2 (C-
5A), 76.5 (C-7D), 75.5 (2 C, C-2A, C-2C), 75.2 (C-4A), 74.3 (2 C, C-
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York

1354
P. K. Mandal, A. K. Misra
PAPER
(2) (a) Holmgren, J.; Svennerholm, A. M.; Lindblad, M. Infect.
Immun. 1983, 39, 147. (b) Chaturvedi, P.; Warren, C. D.;
Altaye, M.; Morrow, A. L.; Ruiz-Palacios, G.; Pickering, L.
K.; Newburg, D. S. Glycobiology 2001, 11, 365.
(7) Nakamura, T.; Urashima, T. Trends Glycosci. Glycotechnol.
2004, 16, 135.
(8) Meijer, A.; Ellervik, U. J. Org. Chem. 2004, 69, 6249.
(9) Yashunsky, D. U.; Higson, A. P.; Ross, A. J.; Nikolaev, A.
V. Carbohydr. Res. 2001, 336, 243.
(10) Berces, A.; Whitfield, D. M.; Nukada, T.; Santos, I. Z.;
Obuchowska, A.; Krepinsky, J. J. Can. J. Chem. 2004, 82,
1157.
(11) Fried, J.; Walz, D. E. J. Am. Chem. Soc. 1949, 71, 140.
(12) Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H.; Kiso, M.
Carbohydr. Res. 1991, 212, 277.
(13) (a) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331. (b) Konradsson, P.;
Udodong, U. E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31,
4313.
(14) Agnihotri, G.; Misra, A. K. Tetrahedron Lett. 2006, 47,
3653.
(15) Schmidt, R. R.; Jung, K.-H. Preparative Carbohydrate
Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York,
1997, 283–312.
(16) (a) Hasegawa, A.; Kiso, M. In Carbohydrates – Synthetic
Methods and Applications in Medicinal Chemistry; Ogura,
H.; Hasegawa, A.; Suami, T., Eds.; Wiley: New York, 1992,
243–266. (b) Hasegawa, A.; Kiso, M. In Preparative
Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel
Dekker: New York, 1997, 357–379. (c) Boons, G.-J.;
Demchenko, A. V. Chem. Rev. 2000, 100, 4539. (d) Kanie,
O.; Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1988, 7,
501. (e) Paulsen, H.; Tietz, H. Angew. Chem., Int. Ed. Engl.
1982, 21, 927. (f) Hasegawa, A.; Nagahama, T.; Ohki, T.;
Hotta, K.; Ishida, H.; Kiso, M. J. Carbohydr. Chem. 1991,
10, 493. (g) Kiso, M.; Ando, K.; Inagaki, H.; Ishida, H.;
Hasegawa, A. Carbohydr. Res. 1995, 272, 159. (h) Castro-
Palomino, J. C.; Ritter, G.; Fortunato, S. R.; Reinhardt, S.;
Old, L. J.; Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1997,
36, 1998. (i) Iida, M.; Endo, A.; Fujita, S.; Numata, M.;
Sugimoto, M.; Nunomura, S.; Ogawa, T. J. Carbohydr.
Chem. 1998, 17, 647.
(c) Gothefors, L.; Olling, S.; Winberg, J. Acta Pediatr.
Scand. 1975, 64, 807. (d) Andersson, B.; Porras, O.;
Hanson, L. A.; Lagergard, T.; Svanborg-Eden, C. J. Infect.
Dis. 1986, 153, 232. (e) Coppa, G. V.; Gabrielli, O.; Pierani,
P.; Catassi, C.; Carlucci, A.; Giorgi, P. Pediatrics 1993, 91,
637. (f) Crane, J. K.; Azar, S. S.; Stam, A.; Newburg, D. S.
J. Nutr. 1994, 124, 2358. (g) Gyorgy, P.; Jeanloz, R. W.;
von Nicolai, H.; Zilliken, F. Eur. J. Biochem. 1974, 43, 29.
(h) Ofek, I.; Beachey, E. H. Adv. Intern. Med. 1980, 25, 503.
(i) Laegreid, A.; Otnaess, A. B. K.; Fuglesang, J. Pediatr.
Res. 1986, 20, 416. (j) Zopf, D.; Roth, S. Lancet 1996, 347,
1017. (k) Cravioto, A.; Tello, A.; Villafan, H.; Ruiz, J.;
Del Vedovo, S.; Neeser, J.-R. J. Infect. Dis. 1991, 163,
1247. (l) Newburg, D. S.; Pickering, L. K.; McCluer, R. H.;
Cleary, T. C. J. Infect. Dis. 1990, 162, 1075.
(3) Coppa, G. V.; Gabrielli, O.; Giorgi, P.; Catassi, C.;
Montanari, M. P.; Varaldo, P. E.; Nichols, B. L. Lancet
1990, 335, 569.
(4) (a) Feizi, T. Nature 1985, 314, 53. (b) Wieruszeski, J. M.;
Chekkor, A.; Bouquelet, S.; Montreuil, J.; Strecker, G.;
Peter-Katalinic, J.; Egge, H. Carbohydr. Res. 1985, 137,
127. (c) Strecker, G.; Fievre, S.; Wieruszeski, J. M.;
Michalski, J. C.; Montreuil, J. Carbohydr. Res. 1992, 226,
1. (d) Dua, V. K.; Goso, K.; Dube, V. E.; Bush, C. A. J.
Chromatogr. 1985, 328, 259. (e) Yamashita, K.; Mizuochi,
T.; Kobata, A. Methods Enzymol. 1982, 83, 105.
(5) (a) Gopal, P. K.; Gill, H. S. Br. J. Nutr., Suppl. 1 2000, 84,
S69. (b) Sharon, N.; Ofek, I. Glycoconj. J. 2000, 17, 659.
(c) Boedeker, E. C. Gastroenterology 1982, 83, 489.
(6) (a) Urashima, T.; Saito, T.; Nakamura, T.; Messer, M.
Glycoconj. J. 2001, 18, 357. (b) Messer, M.; Urashima, T.
Trends Glycosci. Glycotechnol. 2002, 14, 153.
(c) Guerardel, Y.; Morelle, W.; Plancke, Y.; Lemoine, J.;
Strecker, G. Carbohydr. Res. 1999, 320, 230. (d) Saksena,
R.; Deepak, D.; Khare, A.; Sahai, R.; Tripathi, L. M.;
Srivastava, V. M. L. Biochem. Biophys. Acta 1999, 1428,
433. (e) Chaturvedi, P.; Sharma, C. B. Biochim. Biophys.
Acta 1988, 967, 115. (f) Urashima, T.; Bubb, W. A.;
Messer, M.; Tsuji, Y.; Taneda, Y. Carbohydr. Res. 1994,
262, 173.
(17) Pearlman, W. M. Tetrahedron Lett. 1967, 8, 1663.
Synthesis 2009, No. 8, 1348–1354 © Thieme Stuttgart · New York