First synthesis of the immunodominant β-galactofuranose-containing tetrasaccharide present in the cell wall of Aspergillus fumigatus

Article abstract of DOI:10.1016/j.carres.2004.10.019

β-Galf-(1→5)-β-Galf-(1→6)-α-Manp-(1→6) -α-Manp, the immunodominant epitope in the cell-wall galactomannan of Aspergillus fumigatus, was synthesized for the first time as its allyl glycoside. The key disaccharide glycosyl donor, 2,3,5,6-tetra-O-benzoyl-β-

Full text of DOI:10.1016/j.carres.2004.10.019

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 25–30
First synthesis of the immunodominant b-galactofuranose-containing
tetrasaccharide present in the cell wall of Aspergillus fumigatus
Mingkun Fu, Guohua Zhang and Jun Ning*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871, Beijing 100085, China
Received 7 September 2004; accepted 20 October 2004
Available online 21 November 2004
Abstract—b-Galf-(1!5)-b-Galf-(1!6)-a-Manp-(1!6)-a-Manp, the immunodominant epitope in the cell-wall galactomannan of
Aspergillus fumigatus, was synthesized for the first time as its allyl glycoside. The key disaccharide glycosyl donor, 2,3,5,6-
tetra-O-benzoyl-b-D-galactofuranosyl-(1!5)-2-O-acetyl-3,6-di-O-benzoyl-b-D-galactofuranosyl trichloroacetimidate (10), was
constructed by 5-O-glycosylation of 1,2-O-isopropylidene-3,6-di-O-benzoyl-a-D-galactofuranose (4) with 2,3,5,6-tetra-O-benzoyl-
b-D-galactofuranosyl trichloroacetimidate (5), followed by 1,2-O-deacetonation, acetylation, selective 1-O-deacetylation, and
trichloroacetimidation. The target tetrasaccharide 16 was obtained by the condensation of allyl 2,3,4-tri-O-benzoyl-a-D-mannopy-
ranosyl-(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (14) as glycosyl acceptor with the disaccharide glycosyl donor 10,
followed by deprotection.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Oligosaccharide; Galactofuranose; Synthesis
1. Introduction
illus cell wall, considering its high immunogenicity.5
6c
Notermans and Soentoro6a and Latge have identified
the diagnostic potential of the Aspergillus cell-wall poly-
´
Aspergillus fumigatus, a fungus, is the major causative
agent of several respiratory tract-related diseases such
as invasive aspergillosis, airway cavity colonization,
and allergic manifestations.1 Invasive aspergillosis is a
leading cause of death among those treated for hemato-
logical malignancy and those receiving a solid organ
transplant, especially of the lung,2 due to the infection
in allogeneic hematopoietic stem-cell-transplant recipi-
ents.3 The high mortality of invasive aspergillosis is
due partly to difficulties in timely diagnosis because sig-
nals and symptoms are often nonspecific. For instance,
only at a late stage of invasive aspergillosis did cultures
of respiratory specimens become positive.4 Hence, in the
search for a potential structure that could be helpful in
the diagnosis of the different forms of aspergillosis,
much attention has been paid to the study of the Asperg-
saccharides and glycoproteins. Barreto-Bergter and
6b
co-workers,6d,e Latge et al., Bennnet et al.,7 and
´
Notermans et al.8 have studied the structure of galacto-
mannans from the Aspergillus cell wall. Later on,
through extensive exploration,9 it was found that the
galactomannans from Aspergillus could be regarded as
a marker to indicate invasive aspergillosis to improve
timely diagnosis. That is to say, when galactomannans10
released by Aspergillus are detected in the serum or plas-
ma of patients, a prompt diagnosis can be used to reduce
the high ratio of mortality of invasive aspergillosis. For
instance, in around two-thirds of the patients, galacto-
mannan could be detected at a mean of 8days before
diagnosis by other means.11 Recent studies12 shown that
tetra- and hexasaccharide, that is, b-Galf-(1!5)-b-Galf-
(1!6)-a-Manp-(1!6)-Man and b-Galf-(1!5)-b-Galf-
(1!5)3-b-Galf-(1!6)-Man, fragments of the galacto-
mannan, are the immunodominant epitopes of the
galactomannan. The tetrasaccharide seems to be the
*
Corresponding author. Fax: +86 10 62923563; e-mail: jning@
mail.rcees.ac.cn
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.10.019

26
M. Fu et al. / Carbohydrate Research 340 (2005) 25–30
pound 6 was prepared by coupling compound 4 and
1 5
β
1 6
β
1 6
Galf
Galf
Manp
Manp
α
2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl trichloro-
acetimidate (5)16 in the presence of a catalytic amount
of TMSOTf in excellent yield (93%) (Scheme 1). Both
1H NMR and TLC were employed to detect the (1!5)-
linked disaccharide 6. The structure of 6 was confirmed
by the 1H NMR data. The characteristic resonances
due to the anomeric protons H-1 and H-10 were located
as a doublet at 5.93ppm with J1,2 = 4.1Hz and as a singlet
at 5.69ppm, respectively. The deisopropylidenation of 6
in 10:1 CHCl3–CF3COOH (v/v) at room tempera-
ture gave 2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-
(1!5)-3,6-di-O-benzoyl-b-D-galactofuranose (7), which
after purification was treated with Ac2O and pyridine at
room temperature to afford 2,3,5,6-tetra-O-benzoyl-b-
D-galactofuranosyl-(1!5)-2-O-acetyl-3,6-di-O-benzoyl-
b-D-galactofuranose (8). The diacetate 8 was selectively
deacetylated at the anomeric position with benzylamine
in THF in high yield to give the corresponding 2,3,5,6-tet-
ra-O-benzoyl-b-D-galactofuranosyl-(1!5)-2-O-acetyl-
3,6-di-O-benzoyl-b-D-galactofuranose (9). Subsequent
reaction of 9 with CCl3CN–K2CO3 in CH2Cl2 afforded
the 2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-(1!5)-
2-O-acetyl-3,6-di-O-benzoyl-b-D-galactofuranosyl tri-
chloroacetimidate (10). The structure of 10 was confi-
rmed by 1H NMR spectral analysis as follows: d 8.63 (s,
1H, CNHCCl3), d 6.45 (s, 1H, H-10), d 5.74 (s, 1H,
H-1).
1
1 2
α
1 2
α
1 6
2
α
1
Manp
Manp
Manp
Manp
α
α
n
3
β
1
6
1
β
Galf
Galf
p
5
5
1
β
β
1
n>10
P>3
Galf
Galf
2
Figure 1. Structures of the immunodominant tetrasaccharide 1 of the
cell wall galactomannan 2 of Aspergillus fumigatus.
minimum structure required for the immunodominant
epitope in the mycelial cell wall of A. fumigatus (Fig. 1).
Providing enough sample is a basic condition for de-
tailed studies on a compoundꢀs fundamental biochemi-
cal properties and possible biological functions.
However, in the carbohydrate field, these efforts are
often frustrated by the difficulty of synthesizing saccha-
rides. Synthesis of complex oligosaccharide sequences
containing two to six sugar units, both in solution and
on solid phase, presents a major challenge in organic
chemistry.13 Unlike peptides and nucleic acids, oligosac-
charides are typically branched rather than linear. The
monosaccharide units can be connected by a or b link-
ages. Furthermore oligosaccharide synthesis requires
multiple selective protection and deprotection steps.
Although over the past few decades considerable pro-
gress14 has been made in this field, there still is no gen-
eral route for oligosaccharide synthesis.
Study on the synthesis of the immunodominant
oligosaccharide present in the mycelial cell wall of
A. fumigatus is important. On the one hand, sufficient
quantities of the sample can be provided by this means
for its immunoassay studies in detail; on the other hand,
it could be used to further elucidate the molecular struc-
ture responsible for monitoring invasive aspergillosis.
These, together with the fact that synthesis of the tetra-
saccharide has never been done so far, prompted us to
synthesize b-Galf-(1!5)-b-Galf-(1!6)-a-Manp-(1!6)-
a-Manp as its allyl glycoside 16.
6-O-Acetyl-2,3,4-tri-O-benzoyl-a-D-mannopyranosyl
trichloroacetimidate (11) was prepared from D-mannose
according to the reported procedure.17 Glycosylation of
11 with allyl alcohol in the presence of TMSOTf, followed
by removal of the 6-O-acetyl group in MeOH containing
1% HCl, gave allyl 2,3,4-tri-O-benzoyl-a-D-mannopyr-
anoside 12 in 81% yield (Scheme 2). Condensation of 11
˚
and 12 with TMSOTf as catalyst and 4A molecular sieves
in CH2Cl2 at ꢀ20ꢁC, followed by removal of acetyl group
of allyl 6-O-acetyl-2,3,4-tri-O-benzoyl-a-D-mannopyrano-
syl-(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (13),
afforded allyl 2,3,4-tri-O-benzoyl-a-D-mannopyranosyl-
(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (14).
The 1H NMR spectra of 14 showed two characteristic sig-
nals for H-1 at 5.16ppm with J1,2 = 1.4Hz and H-10 at
5.14ppm with J1,2 = 1.3Hz, respectively.
2. Results and discussion
With the glycosyl donor and acceptor 10 and 14 in
hand, construction of the target compound was readily
carried out. As shown in Scheme 3, the fully protected
tetrasaccharide, allyl l,3,5,6-tetra-O-benzoyl-b-D-galac-
tofuranosyl-(1!5)-3,6-di-O-benzoyl-2-O-acetyl-b-D-gal-
actofuranosyl-(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopy-
ranosyl-(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyrano-
side 15, was obtained by coupling 10 with 14 in 85%
3,6-Di-O-benzoyl-1,2-O-isopropylidene-a-D-galactofur-
anose (4) is an important synthetic intermediate in our
synthesis, which was easily obtained through selective
benzoylation of 3-O-benzoyl-1,2-O-isopropylidene-a-D-
galactofuranose (3)15a with BzCl in pyridine at 0ꢁC in
82% yield. Although a similar disaccharide was published
in 1990,15b our method for preparing the novel disc-
acharide, 2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-
(1!5)-3,6-di-O-benzoyl-1,2-O-isopropylidene-a-D-gal-
actofuranose (6), is simple and is more efficient. Com-
1
yield. The H NMR data of 15 contained structurally
characteristic information: for instance, one acetyl sig-
nal (d 1.86), one allyl signal (d 5.52–5.32), and four H-
1 signals (d 5.74, 5.22, 5.20, 5.18). The 13C NMR spec-

M. Fu et al. / Carbohydrate Research 340 (2005) 25–30
27
O
O
OBz
OBz
a
HO
BzO
O
O
O
OH
O
OH
4
3
O
OBz
NH
OCCCl3
BzO
O
O
O
O
c
b
OBz
BzO
O
4
+
OBz
OBz
BzO
OBz
OBz
OBz
5
6
OR1
O
O
OBz
OH
OBz
BzO
BzO
OR2
O
OH
d
O
O
OBz
O
OBz
BzO
BzO
OBz
OBz
OBz
8 R1 = R2 = Ac
OBz
e
f
7
9 R1 = Ac, R2 = H
10 R1 = OAc, R2 = OCNHCCl3
˚
Scheme 1. Reagents and conditions: (a) PhCOCl (1.2equiv), pyridine, 0ꢁC, 6h; (b) TMSOTf (cat), CH2Cl2, 4A MS powder, ꢀ20ꢁC to rt, 1.5h, 93%;
(c) 10:1 HCCl3–CFCOOH, rt, 2h; (d) Ac2O, pyridine, rt, 12h; (e) PhCH2NH2 (4.0equiv), THF, rt, 24h; (f) CCl3CN (2equiv), CH2Cl2, K2CO3, rt,
5h.
second allyl signal (d 5.30–5.25, dd), a third allyl signal
HO
AcO
BzO
OBz
O
OBz
O
(d 5.21–5.18, dd), and four H-1 signals (d 5.14, 4.95,
4.81, 4.80). In addition, the chemical shifts of the ano-
meric carbons of 16 revealed by its 13C NMR spectrum
were at 107.71, 107.08, 99.57, 99.27ppm, confirming the
structure of 16.
In summary, an efficient synthesis of the immuno-
dominant tetrasaccharide 16 present in the cell-wall
galactomannan of A. fumigatus was achieved for the first
time. This would promote the studies on fundamental
biochemical properties and biological functions about
this oligosaccharide, and consequently promote the
treatment of diseases caused by this pathogen.
a
BzO
BzO
BzO
OAllyl
OCCCl3
NH
12
11
11
b
R1O
OBz
O
BzO
BzO
O
OBz
O
BzO
BzO
OAllyl
13 R1 = Ac
14 R1 = H
c
Scheme 2. Reagents and conditions: (a) (i) allyl alcohol (2equiv),
TMSOTf, CH2Cl2, rt; (ii) 1% CH3COCl in CH2Cl2–CH3OH, rt, 10–
3. Experimental
˚
12h; (b) TMSOTf (cat), CH2Cl2, 4A MS powder, ꢀ20ꢁC to rt, 1.5h;
3.1. General methods
(c) 1% CH3COCl in CH2Cl2–CH3OH, rt, 10–12h.
Melting points were determined with a ꢁMel-Tempꢀ appa-
ratus. Optical rotations were measured at 25ꢁC in the sta-
trum of 15 gave four signals for C-1 (d 106.45, 105.94,
97.69, 96.85). Deprotection of 15 in ammonia-saturated
methanol yielded the target allyl b-D-galactofuranosyl-
(1!5)-b-D-galactofuranosyl-(1!6)-a-D-mannopyrano-
syl-(1!6)-a-D-mannopyranoside (16). The signals of the
1H NMR spectrum of 16 contained structurally charac-
teristic information: one allyl signal (d 5.92–5.84, m), a
1
ted solvent. H NMR (400Hz) and 13C NMR (100Hz)
spectra were recorded in CDCl3 solutions at room tem-
perature unless otherwise specified. Chemical shift (d)
values are given in ppm; coupling constants (J) are
in Hertz. Mass spectra were recorded on an Autospec
mass spectrometer using the electrospray-ionization

28
M. Fu et al. / Carbohydrate Research 340 (2005) 25–30
O
O
OBz
OBz
O
BzO
BzO
BzO
OAc
O
10
O
+
14
OBz
O
O
BzO
OBz
BzO
BzO
OAllyl
OBz
OBz
15
b
O
O
OH
OH
O
HO
HO
HO
OH
O
O
O
OH
O
OH
HO
HO
HO
OH
OAllyl
OH
16
Scheme 3. Reagents and conditions: (a) TMSOTf (cat), CH2Cl2, 4A MS powder, ꢀ20ꢁC to rt, 1.5h; (b) satd aq NH3–MeOH, rt, 24h.
˚
technique. Thin-layer chromatography (TLC) was per-
formed on silica gel HF254 with detection by charring
with 30% (v/v) H2SO4 in MeOH or in some cases by a
UV detector. Column chromatography was conducted by
elution of a column (10 · 240mm, 18 · 300mm, 35 ·
400mm) of silica gel (100–200 mesh) with EtOAc–petro-
leum ether (60–90ꢁC) as the eluent. Solutions were con-
centrated at <60ꢁC under diminished pressure. Dry solvents
were distilled over CaH2 and stored over molecular sieves.
molecular sieves (4g) at room temperature under an
atmosphere of nitrogen for 20min. Then the reaction
mixture was cooled to ꢀ20ꢁC, and TMSOTf (20lL,
0.1mmol) was added. After 30min, the mixture was al-
lowed to rise to room temperature. The reaction mixture
was stirred for further 1h, at the end of which time TLC
(2.5:1 petroleum ether–EtOAc) indicated that the reac-
tion was complete. The reaction mixture was neutralized
with Et3N and filtered, and the filtrate was concentrated.
The resultant residue was subjected to column chroma-
tography with 2.5:1 petroleum ether–EtOAc as eluent to
afford the disaccharide 6 (11.2g, 89%): [a]D +12.7 (c 0.5,
3.2. Preparation of 1,2-O-isopropylidene-3,6-di-O-benz-
oyl-a-D-galactofuranose (4)
1
CHCl3); H NMR (400MHz, CDCl3): d 8.10–7.22 (m,
To a solution of 3 (5.0g, 15.4mmol) in dry pyridine
(30mL) was added dropwise a solution of benzoyl chlo-
ride (2.0mL, 17mmol) in dry CH2Cl2 (10mL) at ꢀ5ꢁC
over 30min. Then the mixture was warmed to 0ꢁC
and stirred until TLC (4:1 petroleum ether–EtOAc) indi-
cated that the reaction was complete. Then the mixture
was poured into water and extracted with CH2Cl2. The
organic layer was washed with 1N HCl and satd aq
NaHCO3. The solution was concentrated under vac-
uum. The residue was purified by flash chromatography
(2:1 petroleum ether–EtOAc) to give 4 as a syrup (4.8g,
90 %): [a]D ꢀ10.2 (c 0.8, CHCl3) (lit.18 ꢀ15.2 (c 1.00)).18
30H, 6PhH), 6.21 (m, 1H, H-5), 5.93 (d, 1H,
J = 4.1Hz, H-1), 5.78 (s, 1H, H-3), 5.69 (s, 1H, H-10),
5.63 (d, 1H, J = 4.1Hz, H-30), 5.56 (s, 1H, H-20), 5.11
(m, 1H, H-4), 4.88 (dd, 1H, J = 4.4, 11.6Hz, H-60),
4.76–4.69 (m, 3H), 4.68 (m, 1H), 4.59 (dd, 1H, J = 5.2,
9.2Hz, H-6), 4.37 (m, 1H), 1.68 (s, 3H, CH3), 1.28 (s,
3H, CH3). Anal. Calcd for C57H50O17Æ0.5H2O: C,
67.38; H, 5.06. Found: C, 67.40; H, 5.05.
3.4. 2,3,5,6-Tetra-O-benzoyl-b-D-galactofuranosyl-
(1!5)-2-O-acetyl-3,6-di-O-benzoyl-b-D-galactofuranosyl
trichloroacetimidate (10)
3.3. 2,3,5,6-Tetra-O-benzoyl-b-D-galactofuranosyl-
(1!5)-3,6-di-O-benzoyl-1,2-O-isopropylidene-a-D-galac-
tofuranose (6)
To a solution of 8 (5.9g, 5.64mmol) and benzylamine
(22.6mmol) in a solution of THF (80mL) was kept in
the dark at room temperature for 24h. At the end of this
time, TLC (3:1 petroleum ether–EtOAc) showed that
the reaction was complete, and the solution was concen-
trated. The resultant residue without purification was
A solution of 4 (4.2g, 9.9mmol) and 5 (8.0g, 10.8mmol)
˚
in dry CH2Cl2 (60mL) was stirred with activated 4A

M. Fu et al. / Carbohydrate Research 340 (2005) 25–30
29
˚
dissolved in dry CH2Cl2 (50mL), and then trichloro-
acetonitrile (2.6mL, 12.4mmol) and anhydrous K2CO3
(3g, 21.8mmol) were added. The reaction mixture was
stirred at room temperature for 12h, and the solid mate-
rial was filtered off. Concentration of the filtrate, fol-
lowed by purification on a silica gel column with 3:1
petroleum ether–EtOAc as eluent, gave the disaccharide
vated 4A molecular sieves (2g) at room temperature un-
der an atmosphere of nitrogen for 20min. Then the
reaction mixture was cooled to ꢀ20ꢁC, and TMSOTf
(12lL, 0.06mmol) was added. After 30min, the mixture
was allowed to rise to room temperature. The reaction
mixture was stirred for further 1h, at the end of which
time TLC (2:1 petroleum ether–EtOAc) indicated that
the reaction was complete. The reaction mixture was
neutralized with Et3N and filtered, and the filtrate was
concentrated under vacuum. Then the resultant residue
was directly treated with 1% CH3COCl in CH2Cl2–
CH3OH at room temperature. The solution was stirred
at room temperature until TLC (1.5:1 petroleum
ether–EtOAc) showed that the starting material disap-
peared. The solution was neutralized with Et3N and
concentrated under vacuum. The resultant residue was
subjected to column chromatography with 1.5:1 petro-
leum ether–EtOAc as eluent to afford the disaccharide
1
donor 10 (4.6g, 90%): [a]D +15.2 (c 0.8, CHCl3); H
NMR (400MHz, CDCl3): d 8.63 (s, 1H, NH), 8.06–
7.24 (m, 30H, 6PhH), 6.45 (s, 1H, H-10), 6.10 (m, 1H,
H-50), 5.75 (d, J = 4.2Hz, H-30), 5.72 (s, 1H, H-1),
5.62 (d, 2H, J = 4.2Hz, H-3), 5.60 (s, 1H, H-20), 5.48
(s, 1H, H-2), 5.00 (m, 1H, H-40), 4.81 (dd, 1H, J = 3.6,
12Hz, H-60), 4.73 (dd, 1H, J = 3.6, 12Hz, H-6), 4.71–
4.64 (m, 3H), 4.61–4.59 (m, 1H), 1.99 (s, 3H, CH3CO).
Anal. Calcd for C58H48Cl3NO18: C, 60.40; H, 4.19.
Found: C, 60.12; H, 4.22.
1
3.5. Allyl 2,3,4-tri-O-benzoyl-a-D-mannopyranoside (12)
A solution of 11 (4.3g, 6.4mmol) in dry CH2Cl2 (40mL)
acceptor 14 (4.6g, 90%): [a]D ꢀ72.3 (c 0.8, CHCl3); H
NMR (400MHz, CDCl3):
d 8.17–7.25 (m, 30H,
6PhH), 6.06 (m, 1H, CH2@CH–CH2O), 6.04 (dd, 1H,
J = 3.2, 10Hz, H-30), 6.02 (dd, 1H, J = 10.1Hz, H-40),
5.94 (dd, 1H, J = 3.2, 10Hz, H-3), 5.80 (dd, 1H,
J = 10.1Hz, H-4), 5.74 (m, 2H, H-2, H-20), 5.48 (dd,
1H, J = 1.2, 17.2Hz, CH2@CH–CH2O), 5.33 (dd, 1H,
J = 1.2, 17.2Hz, CH2@CH–CH2O), 5.16 (d, 1H,
J = 1.6Hz, H-10), 5.14 (d, 1H, J = 1.6Hz, H-1), 4.44–
4.21 (m, 2H, CH2@CH–CH2O), 4.40–4.39 (m, 1H, H-
50), 4.08–4.00 (m, 2H, H-60, H-5), 3.76 (dd, 1H,
J = 1.9, 10.9Hz, H-60), 3.61 (dd, 1H, J = 1.9, 10.9Hz,
H-6), 3.53 (dd, 1H, J = 4.3, 12.9Hz, H-6). Anal. Calcd
for C57H50O17: C, 67.99; H, 5.00. Found: C, 67.76; H,
4.83.
˚
was stirred with activated 4A molecular sieves (1.5g) at
room temperature under an atmosphere of nitrogen for
20min. After the reaction mixture was cooled to ꢀ20ꢁC,
allyl alcohol (0.77mL, 12.8mmol) and TMSOTf (20lL,
0.1mmol) was added. After 30min, the mixture was al-
lowed to rise to room temperature, and the reaction
mixture was stirred until TLC (2.5:1 petroleum ether–
EtOAc) indicated that the reaction was complete. The
reaction mixture was neutralized with Et3N and filtered,
and the filtrate was concentrated under vacuum. Then
the resultant residue was directly treated with 1%
CH3COCl in CH2Cl2–CH3OH at room temperature.
The solution was stirred at room temperature until
TLC (2:1 petroleum ether–EtOAc) showed that the
starting material disappeared. The solution was neutral-
ized with Et3N and concentrated under vacuum. The
residue was purified by flash chromatography with 2:1
petroleum ether–EtOAc as eluent to afford 12 (2.9g,
3.7. Allyl 2,3,5,6-tetra-O-benzoyl-b-D-galactofuranosyl-
(1!5)-3,6-di-O-benzoyl-2-O-acetyl-b-D-galactofurano-
syl-(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranosyl-
(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (15)
1
92%): [a]D ꢀ108.2 (c 0.8, CHCl3); H NMR (400MHz,
A solution of 10 (2.9g, 2.52mmol) and 14 (2.5g,
2.52mmol) in dry CH2Cl2 (50mL) was stirred with acti-
˚
vated 4A molecular sieves (2g) at room temperature
CDCl3): d 8.12–7.24 (m, 15H, 3PhH), 6.02 (dd, 1H,
J = 3.6, 10.1Hz, H-3), 5.96 (m, 1H, CH2@CH–CH2O),
5.86 (dd, 1H, J = 10.1Hz, H-4), 5.70 (dd, 1H, J = 1.2,
3.0Hz, H-2), 5.35 (dd, 2H, J = 10.4, 36.3Hz,
CH2@CH–CH2O), 5.17 (d, 1H, J = 1.2Hz, H-1), 4.32
(dd, 1H, J = 5.2, 12.9Hz, H-6a), 4.15 (dd, 1H, J = 5.2,
12.9Hz, H-6), 4.10 (m, 1H, H-5), 3.85 (m, 1H,
CH2@CH–CH2O), 3.78 (dd, 1H, J = 3.7, 12.8Hz,
CH2@CH–CH2O). Anal. Calcd for C30H28O9: C,
67.66; H, 5.30. Found: C, 67.35; H, 5.23.
under an atmosphere of nitrogen for 20min. Then the
reaction mixture was cooled to ꢀ20ꢁC, and TMSOTf
(15lL, 0.07mmol) was added. After 30min, the mixture
was allowed to rise to room temperature. The reaction
mixture was stirred for further 1h, at the end of which
time TLC (2.5:1 petroleum ether–EtOAc) indicated that
the reaction was complete. The reaction mixture was
neutralized with Et3N and filtered, and the filtrate was
concentrated. The resultant residue was subjected to
the column chromatography with 2.5:1 petroleum
ether–EtOAc as eluent to afford the tetrasaccharide 15
(4.9g, 92%): [a]D ꢀ64.5 (c 0.8, CHCl3); 1H NMR
(400MHz, CDCl3): d 8.00–7.23 (m, 60H, 12PhH), 6.13
(dd, 1H, J = 10.2Hz, H-40), 6.12–6.04 (m, 2H, H-5000,
3.6. Allyl 2,3,4-tri-O-benzoyl-a-D-mannopyranosyl-
(1!6)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (14)
A solution of 11 (3.6g, 5.25mmol) and 12 (2.8g,
5.25mmol) in dry CH2Cl2 (50mL) was stirred with acti-

30
M. Fu et al. / Carbohydrate Research 340 (2005) 25–30
CH2@CHCH2O), 6.07 (dd, 1H, J = 4, 10.2Hz, H-30),
6.01 (dd, 1H, J = 10.1Hz, H-4), 5.98 (dd, 1H, J = 3.4,
10.1Hz, H-3), 5.78 (m, 2H, H-2, H-20), 5.74 (s, 1H,
H-1000), 5.66 (d, J = 5.2Hz, H-3000), 5.65 (d, J = 4.2Hz,
H-30), 5.63 (s, 1H, H-2000), 5.50 (dd, J = 1.3, 11.0
Hz, CH2@CHCH2O), 5.34 (dd, J = 1.3, 11.0Hz,
CH2@CHCH2O), 5.33 (s, 1H, H-20), 5.22 (s, 1H, H-
10), 5.20 (d, 1H, J = 1.3Hz, H-10), 5.18 (d, 1H,
J = 1.4Hz, H-1), 5.00 (dd, J = 4.2Hz, H-4), 4.80 (dd,
1H, J = 3.7, 12.0Hz, H-6000), 4.67 (dd, 1H, J = 7.8,
12.2Hz, H-60), 4.63–4.59 (m, 2H), 4.89–4.42 (m, 3H),
4.41–4.26 (m, 2H, CH2@CHCH2O), 4.24–4.21 (m, 1H,
H-5), 4.13 (dd, 1H, J = 5.0, 11.1Hz, H-6), 3.79–3.76
(m, 2H, 2 H-60), 3.64 (dd, 1H, J = 5.0, 11.1Hz, H-6),
1.86 (s, 3H, CH3CO); 13C NMR (100MHz, CDCl3): d
169.53, 166.04, 166.01, 165.72, 165.66, 165.61, 165.47,
165.39, 165.21, 165.16, 118.46, 106.45, 105.94, 97.69,
96.85, 82.30, 82.13, 81.55, 81.39, 77.88, 77.38, 77.06,
76.99, 76.74, 73.09, 70.65, 70.59, 70.39, 70.29, 70.05,
69.76, 68.86, 66.97, 66.63, 65.93, 65.09, 63.91, 20.48.
Anal. Calcd for C113H96O34: C, 67.93; H, 4.85. Found:
C, 67.72; H, 4.80.
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1
[a]D ꢀ7.9 (c 0.5, H2O); H NMR (400MHz, D2O): d
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5.92–5.84 (m, 1H), 5.30–5.25 (dd, 1H, J = 17.1Hz),
5.21–5.18 (dd, 1H, J = 10.4Hz), 5.14 (d, 1H,
J = 2.0Hz, 1H-1), 4.95 (s, 1H, 1H-1), 4.81 (d, 1H,
J = 1.6Hz, H-1), 4.80 (s, 1H, H-1); 13C NMR
(100MHz, D2O): d 133.35, 118.49, 107.71, 107.08,
99.57, 99.27, 82.62, 81.80, 81.26, 81.01, 76.68,
76.53, 75.87, 71.66, 70.91, 70.85, 70.55, 70.06, 69.94,
68.34, 66.62, 65.74, 62.83, 61.08. Anal. Calcd for
C27H46O21: C, 45.89; H, 6.56. Found: C, 45.66; H,
6.40.
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This work was supported by the National Key Project
for Basic Research (2003CB114400), the Beijing Natural
Science Foundation (6021004) and The Ministry of Sci-
ence and Technology (2001AA246014).