Facile syntheses of the hexasaccharide repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

Article abstract of DOI:10.1016/S0968-0896(03)00391-2

Two hexasaccharides, β-D-Xylp-(1→2)-α-D-Manp-(1→3)-[β-D-Xylp-(1→2)-] α-D-Manp-(1→3)-[β-D-GlcpA-(1→2)-]α-D-Manp and β-D-GlcpA-(1→2)-α-D-Manp-(1→3)-[β-D-Xylp-(1→2)-] α-D-Manp-(1→3)-[β-D-Xylp-(1→2)-]α-D-Manp, the repeating unit of the exopolysaccharide from

Full text of DOI:10.1016/S0968-0896(03)00391-2

Bioorganic & Medicinal Chemistry 11 (2003) 4027–4037
Facile Syntheses of the Hexasaccharide Repeating Unit of the
Exopolysaccharide from Cryptococcus Neoformans Serovar A
Jianjun Zhang and Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
Received 25 March 2003; accepted 29 May 2003
Abstract—Two hexasaccharides, b-d-Xylp-(1!2)-a-d-Manp-(1!3)-[b-d-Xylp-(1!2)-]a-d-Manp-(1!3)-[b-d-GlcpA-(1!2)-]a-d-
Manp and b-d-GlcpA-(1!2)-a-d-Manp-(1!3)-[b-d-Xylp-(1!2)-]a-d-Manp-(1!3)-[b-d-Xylp-(1!2)-]a-d-Manp, the repeating unit
of the exopolysaccharide from Cryptococcus neoformans serovar A, were synthesized as their methyl glycosides in a regio- and ste-
reoselective manner.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
assembly of a repeating unit of the exopolysaccharide of
C. neoformans entails inter alia the introduction of a b-
d-glucuronic acid residue at the axially orientated O-2
position of a mannopyranoside moiety.13 The synthesis
of trisaccharide and tetrasaccharide fragments corre-
sponding to structures in capsular polysaccharides of C.
neoformans has been reported14,15 and the synthesis of a
pentasaccharide-the repeating unit of the poly-
saccharide in C. neoformans serovar D has appeared.16
In these syntheses, multiple steps and orthogonal
masking groups were involved making the procedure
rather complex. As a result, it would be difficult to syn-
thesize by the reported methods, the higher oligo-
saccharides — the repeating units of C. neoformans
serotypes A, B, and C. Previously, we have reported the
regio- and stereoselective synthesis of oligosaccharides
with un- or lightly-protected mannose17 and rhamnose18
as the glycosyl acceptors and glycosyl trichloroacet-
imidates as the donors giving satisfactory results. We also
reported in a preliminary communication19 the synthesis
of the hexasaccharide repeating unit of O-deacetylated
GXM of C. neoformans serotype A with 4,6-O-iso-
propylidenated mannose derivatives as the acceptors. We
present herein a newregio- and stereoselective synthesis of
the frame-shifted hexasaccharide and the synthesis in the
precedent communication in detail.
Cryptococcus neoformans is an opportunistic pathogenic
yeast that causes a life-threatening meningoencephalitis
in individuals with an impaired immune system.1 The
incidence of cryptococcosis has increased dramatically
in recent years as a consequence of the AIDS epidemic,
and it is a leading cause of death in patients with
AIDS2À4 C. neoformans is unusual among pathogenic
fungi in that it has a polysaccharide capsule, glucur-
onoxylomannan (GXM).5 GXM is antipathogocytic
and poorly immunogenic, and acapsular strains have
significantly reduced virulence.6 In vitro, GXM inhibits
leukocyte migration,7 enhances HIV infection in human
lymphocytes8 and promotes l-selectin shedding from
neutrophils.9 There are four major serotypes10 for GXM
designated A–D (Scheme 1). All the four serotypes are
composed of a linear a-1,3-linked mannosyl backbone
with b-glucopyranosyluronic acid, b-xylopyranosyl, and
6-O-acetyl substituents.11 Serotype D is the most heavily
O-acetylated and serotype C the least O-acetylated.
However, the O-acetyl groups are not essential for bind-
ing, though they have a significant contribution.5
As part of an ongoing project to synthesize oligo-
saccharides12 existing in cell-wall polysaccharides, we
have reported successful syntheses of 3,6-branched glu-
cans,12a,b,c 3- or 2-branched rhamnans,12d,e 2,6-12f,g and
3,6-branched mannans.12h,i A major problem in the
Results and Discussion
Scheme 2 outlines the newsynthesis of the frame-shifted
hexasaccharide, b-d-Xylp-(1!2)-a-d-Manp-(1!3)-[b-d-
*Corresponding author. Tel.: +86-10-629-36613; fax: +86-10-629-
23563; e-mail: fzkong@mail.rcees.ac.cn
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00391-2

4028
J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
Scheme 1. Model structures of GXM of C. neoformans serotypes A–D.
Xylp-(1!2)-]a-d-Manp-(1!3)-[b-d-GlcpA-(1!2)-]a-d-
Manp. Due to the unstability of methyl ester linkage of
glucuronate residue under either basic or acidic condi-
tions, assembly of the glucuronate unit was arranged at
the end of the reaction series. So, a synthetic route start-
ing from construction of the nonreducing end blocks and
ending with the glucuronosylation was designed and
carried out. Therefore, allyl 3,4,6-tri-O-benzoyl-a-d-
mannopyranoside 1, obtained from rearrangement of
3,4,6-tri-O-benzoyl-1,2-O-allyloxyethylidene-a-d-man-
nopyranose20 followed by selective deacetylation,21 was
chosen as the starting material. Coupling of 1 with 2,3,4
-tri-O-benzoyl-a,b-d-xylopyranosyl trichloroacetimidate
2 afforded (1!2)-linked disaccharide 3. Subsequent
1-O-deallylation with PdCl2 and activation with tri-
chloroacetonitrile22 in the presence of potassium car-
bonate gave the disaccharide donor 4. Condensation of
4 with the acceptor allyl 4,6-O-isopropylidene-a-d-
mannopyranoside 5 selectively afforded the (1!3)-
linked trisaccharide 6 (80.3%). The regioselectivity of
the coupling was confirmed by acetylation of 6 to give 7,
and the 1H NMR spectrum of 7 showed a newly
emerged downfield doublet of doublets at d 5.40 ppm
with J1,2=0.6 and J2,3=3.0 Hz for H-2, compared to
that of 6. Coupling of 6 with 2 gave the tetrasaccharide
8. Because of the presence of benzylidene group, an
attempt for deallylation of 8 with PdCl2 was not suc-
cessful since a complex product was obtained. Thus,
debenzylidenation of 8 with 90% TFA followed by
benzoylation gave fully benzoylated tetrasaccharide 9
gave the pentasaccharide acceptor 15. TMSOTf pro-
moted glucuronosylation of 15 with methyl 2,3,4-tri-O-
acetyl-a-d-glucuronate trichloroacetimidate 16 went
smoothly giving the required hexasaccharide 17 in
satisfactory yield (72.7%). The 13C NMR spectrum of
17 showed all of the characteristic signals such as two
methyl signals (d 54.7 and 52.7 ppm, respectively), 13
benzoyl C¼O signals (d 167.3. 167.2, 166.1, 166.0,
165.8, 165.4, 165.3, 165.3, 165.3, 165.2, 165.2, 165.1,
164.6 ppm), four acetyl C¼O signals (d 171.3, 171.0,
169.7, 169.2 ppm), and six anomeric C signals 102.6,
100.6, 100.6, 99.6, 98.9, 98.2. Deprotection of 17 was
carried out in methanol with MeONa for 96 h, then
water (10 equiv) was added to cleave the methyl ester.
After stirring at room temperature for 2 h, the reaction
mixture was concentrated and purified on a Bio-Gel P2
column (eluent: water), affording the target hex-
asaccharide 18 as a foamy solid. Determination of
JC1,H1 of 18 confirmed mannosyl -linkages
(JC1,H1=176.2, 172.7, and 174.1 Hz), b-xylosyl linkages
(JC1,H1=163.4 and 163.4 Hz), and b-glucopyr-
anosyluronic acid linkage (JC1,H1=166.8 Hz).
Scheme 3 shows synthesis of the another hexasacchar-
ide, b-d-GlcpA-(1!2)-a-d-Manp-(1!3)-[b-d-Xylp-(1!2)-]
a-d-Manp-(1!3)-[b-d-Xylp-(1!2)-]a-d-Manp, with the
glucuronic acid residue at the nonreducing end. For the
same reason of the unstability of methyl ester of glu-
curonate, the synthesis started from construction of the
reducing end blocks, then extended to nonreducing end,
and finally ended with the glucuronosylation. Thus, 2,3-
di-O-acetyl-4,6-O-isopropylidene-a-d-mannopyranosyl
trichloroacetimidate 19 was obtained from selective 4,6-
O-isopropylidenation of mannose,23 acetylation, selec-
tive 1 - O - deacetylation, and followed by tri-
chloroacetimidation. Condensation of 19 with the
acceptor methyl 4,6-O-isopropylidene-a-d-mannopyr-
anoside 20 selectively afforded the (1!3)-linked dis-
accharide 21 (74%). The regioselectivity of the coupling
was confirmed by acetylation of 21 to give 22, and the
1H NMR spectrum of 22 showed a newly emerged
downfield doublet of doublets at d 5.34 ppm with
J1,2=1.5 and J2,3=3.0 Hz for H-2, compared to that of
21. Deacetylation of 21 or 22 in a solution of ammonia
in methanol furnished the disaccharide triol acceptor 23
quantitatively. Again, 3-O-selective glycosylation of 23
with the donor 2-O-acetyl-3,4,6-tri-O-benzoyl-a-d-man-
nopyranosyl trichloroacetimidate 24 yielded (1!3)-
linked trisaccharide 25 (67%). The trisaccharide 25 was
(91.9%). Dallylation of
9
followed by tri-
chloroacetimidation afforded the tetrasaccharide donor
10. Again, 3-O-selective glycosylation of methyl 4,6-O-
isopropylidene-a-d-mannopyranoside 11 with the donor
10 yielded (1!3)-linked pentasaccharide 12. Purifica-
tion of 12 was not easy since an unidentified byproduct
had very similar behavior to 12 in column chromato-
graphy separation. Thus, acetylation of 12 followed by
debenzylidenation yielded the pentasaccharide diol 13
1
(54.3% for three steps) that was easily purified. The H
NMR spectrum of 13 showed a characteristic downfield
doublet of doublets at d 5.24 ppm with J1,2=1.4 and
J2,3=3.2 Hz for H-2, confirming the regioselectivity in
the coupling reaction. Benzoylation of 13 was difficult at
room temperature due to the steric hindrance caused by
xylose residue. So, the benzoylation was carried out in
the presence of catalytic DMAP at 70 ꢀC for 12 h, and
14 was obtained in satisfactory yield (79%). Subsequent
selective deacetylation with CH3COCl–MeOH–CH2Cl2

J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
4029
Scheme 2. Reagents and conditions: (a) TMSOTf (0.01–0.25 equiv), 4 A MS, CH2Cl2, À20 ꢀC, 2–4 h (86% for 3, 80% for 6, 64% for 8, 54% for 12,
73% for 17); (b) (i) PdCl2, HOAc–NaOAc, rt, 10–12 h; (ii) CH2Cl2, CCl3CN (2–3.3 equiv), DBU (0.18–0.2 equiv), rt, 2 h, (90% for 4, 85% for 10);
(c) Ac2O–pyridine, 100%; (d) (i) 90% TFA, rt, 2 h; (ii) BzCl–pyridine, rt, 10 h, 92%; (e) (i) Ac2O–pyridine; (ii) 90% TFA, rt, 2 h; (f) BzCl–pyridine–
DMAP, 70 ꢀC, 12 h, 79%; (g) 2.5% CH3COCl in CH2Cl2–CH3OH, rt, 89%; (h) MeONa–MeOH, rt, 96 h, then water was added, 2 h, 64%.
an ideal acceptor since it contained two free hydroxyl
groups at the positions where the xylosyl residue should
be attached. Thus, TMSOTf promoted dixylosylation of
25 with 2,3,4-tri-O-benzoyl-d-xylopyranosyl tri-
chloroacetimidate 2 was achieved with an ‘inverse
Schmidt’ strategy, that is after formation of monoxy-
lylated tetrasaccharide, additional TMSOTf was added,
and after stirring the reaction mixture for 20 min, fur-
ther 2 equiv of donor 2 was added. Purification of 27 was
not easy since the tetrasaccharide byproducts had very
similar behavior to 27. Thus, deisopropylidenation of 27
in 90% TFA followed by benzoylation with benzoyl
chloride in pyridine gave the protected pentasaccharide
28 (42% for three steps). Selective deacetylation of 28

4030
J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
Scheme 3. Model structures of GXM of C. neoformans serotypes A–D. Reagents and conditions: (a) TMSOTf (0.01–0.05 equiv), 4 A MS, CH2Cl2,
À20 ꢀC, 2–4 h (74% for 21, 67% for 25, 88% for 30); (b) Ac2O–pyridine, 100%; (c) CH3OH saturated with ammonia, rt, 12 h, 100%; (d) 2 (2 equiv),
TMSOTf (0.1 equiv), 4 A MS, CH2Cl2, À0 ꢀC, 0.5 h; then TMSOTf (1.0 equiv), 0.5 h, and 2 (2 equiv) was added, 42%; (e) (i) 90% HOAc, 60 ꢀC, 20
h; (ii) BzCl–pyridine, rt,10 h, 91%; (f) 2% CH3COCl in CH2Cl2–CH3OH, rt, 20 h, 67%; (g) methanol saturated with ammonia, rt, 36 h, then water
(2 equiv) was added, 5 h, 65%.
with 2% acetyl chloride–methanol gave the penta-
saccharide acceptor 29 in 67% yield. Coupling of 29
with methyl 2,3,4-tri-O-acetyl-a-d-glucopyranosylur-
onate trichloroacetimidate 16 went smoothly affording
the protected hexasaccharide in good yield (88%). The
1H and 13C NMR spectra of 30 showed two methyl
signals (d 3.69 and 3.23 ppm, respectively), 13 benzoyl
C¼O signals (d 165.9, 165.9, 165.9, 165.4, 165.4, 165.4,
165.3, 165.2, 165.1, 165.0, 164.9, 164.6, 164.6 ppm), four
acetyl C¼O signals (d 167.0, 168.5, 168.5, 168.3 ppm),
six anomeric C signals (100.9, JC1,H1=175 Hz, Manp;
100.3, JC1,H1=163 Hz, GluAp; 99.9, JC1,H1=164 Hz,
Xylp; 99.5, JC1,H1=163 Hz, Xylp; 98.5, JC1,H1=172 Hz,
Manp; 95.2, JC1,H1=176 Hz, Manp). Deprotection of 30
was carried out in a saturated solution of ammonia in
methanol for 36 h, then water was added to cleave the
methyl ester. After standing at room temperature for 5
h, the reaction mixture was concentrated and purified
on a Bio-Gel P2 column (eluent: water), affording the
target hexasaccharide 31 as a foamy solid.
In summary, efficient syntheses of the hexasaccharide
repeat unit of O-deacetylated GXM of C. neoformans
serotype A with lightly protected mannose derivatives

J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
4031
as the acceptors was achieved. Glycosylation of either
4,6-O-isopropylidenated or 4,6-O-benzylidenated man-
nopyranosides showed excellent 3-O-selectivity. The
strategy presented here also provides a route to the
synthesis of more complex repeating units of GXM of
C. neoformans serotype B and C.
To a solution of 3 (4.88 g, 5 mmol) in 90% acetic acid
(50 mL) containing sodium acetate (1.46 g, 15 mmol)
was added PdCl2 (270 mg, 2.5 mmol), and the mixture
was stirred for 12 h, at the end of which time TLC (2:1
petroleum ether–EtOAc) indicated that the reaction was
complete. The mixture was diluted with dichloro-
methane (150 mL), washed with water and satd aq
sodium bicarbonate. The organic layer was con-
centrated, and the residue was passed through a short
silica gel column with 2:1 petroleum ether–EtOAc as the
eluent to give crude 2,3,4-tri-O-benzoyl-b-d-xylopyr-
anosyl-(1!2)-3,4,6-tri-O-benzoyl-d-mannopyranose as
a syrup. Dried under high vacuum for 2 h, the solid was
dissolved in dichloromethane (30 mL), and CCl3CN (1.0
mL, 10 mmol) and DBU (135 mL, 0.9 mmol) were
added. The reaction mixture was stirred for 2 h, at the
end of which time TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. Concentration
of the reaction mixture, followed by purification of the
crude product on a silica gel column with 3:1 petroleum
ether–EtOAc as the eluent, furnished the disaccharide
donor 4 (4.91 g, 90.9%) as a foamy solid. [a]D À17.5ꢀ (c
1.0, CHCl3); 1H NMR (400 MHz, CDCl3) d 8.73 (s, 1H,
CNHCCL3), 8.19–7.14 (m, 30H, 5Ph), 6.43 (s, 1H, H-1),
6.04 (dd, 1H, J3,4=J4,5=10.1 Hz, H-4), 5.69 (dd, 1H,
J2,3=3.2, J3,4=10.1 Hz, H-3), 5.67 (dd, 1H,
J2,3=J3,4=5.1 Hz, H-30), 5.39 (dd, 1H, J1,2=3.8,
J2,3=5.1 Hz, H-20), 5.12 (m, 1H, H-40), 5.09 (d, 1H,
J1,2=3.8 Hz, H-10), 4.75 (dd, 1H, J1,2=0.6, J2,3=3.2
Hz, H-2), 4.52 (ddd, 1H, J4,5=10.2, J5,6a=6.3,
Experimental
Melting points were determined with a ‘Mel-Temp’
apparatus. Optical rotations were determined at 25 ꢀC
with digital polarimeter. The NMR spectra were recor-
ded in CDCl3 with TMS internal standard or D2O with
ethanol as standard on ARX 400 MHz. Mass spectra
were recorded on an autospec mass spectrometer using
ESI technique to introduce the sample. Elemental ana-
lyses were done on elemental analyzer model 1108 EA.
Thin-layer chromatography (TLC) was performed on
silica gel HF254 with detection by charring with 30%
(v/v) H2SO4 in MeOH or in some cases by a UV detec-
tor. Column chromatography was conducted by elution
of a column (10Â240 mm, 18Â300 mm, 35Â400 mm) of
silica gel (100–200 mesh) with EtOAc-petroleum ether
(60–90 ꢀC) as the eluent. Solutions were concentrated at
<60 ꢀC under diminished pressure. Dry solvents were
distilled over CaH2 and stored over molecular sieves.
0 0
J5,6b=4.6 Hz, H-5), 4.45 (dd, 1H, J4 5 a=2.8,
Allyl
2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl–D-mannopyranoside (3). To
a
J5 a,5 b=12.1 Hz, H-50a), 4.33 (dd, 1H, J4 5 b=3.2,
0 0
0 0
cooled solution (0 ꢀC) of 1 (5.32 g, 10 mmol) and 2 (6.67
g, 1.1 mmol) in anhydrous CH2Cl2 (50 mL) was added
TMSOTf (18 mL, 0.01 mmol). The mixture was stirred
at this temperature for 2 h, and then quenched with
Et3N (two drops). The solvents were evaporated in
vacuo to give a residue, which was purified by silica gel
column chromatography (3:1 petroleum ether–EtOAc)
to give disaccharide 3 (8.41 g, 86.2%) as a foamy solid.
[a]D À23.7ꢀ (c 1.3, CHCl3); 1H NMR (400 MHz,
CDCl3) d 8.16–7.32 (m, 30H, 6Ph), 5.92 (m, 1H,
CH2¼CHCH2O), 5.91 (dd, 1H, J3,4=J4,5=10.2 Hz,
H-4), 5.65 (dd, 1H, J2,3=3.3, J3,4=10.2 Hz, H-3), 5.64
(dd, 1H, J2,3=J3,4=4.9 Hz, H-30), 5.36 (dd, 1H,
J1,2=3.6, J2,3=4.9 Hz, H-20), 5.30–15.18 (m, 2H,
CH2¼CHCH2O), 5.14 (m, 1H, H-40), 4.99 (d, 1H,
J1,2=1.6 Hz, H-1), 4.96 (d, 1H, J1,2=3.6 Hz, H-10), 4.49
(dd, 1H, J1,2=1.6, J2,3=3.3 Hz, H-2), 4.41–4.36 (m, 2H,
H-50), 4.31 (ddd, 1H, J4,5=10.2, J5,6a=6.3, J5,6b=4.6
Hz, H-5), 4.22 (m, 1H, CH2¼CHCH2O), 4.08 (dd, 1H,
J5,6a=6.3, J6a,6b=12.0 Hz, H-6a), 4.02 (m, 1H,
CH2¼CHCH2), 3.37 (dd, 1H, J5,6b=4.6, J6a,6b=12.0
Hz, H-6b); 13C NMR (100 MHz, CDCl3) d 166.1, 166.0,
165.5, 165.4, 165.3, 165.1 (6C, 6PhCO), 133.7–128.1
(PhCO, CH2¼CHCH2O), 118.3 (CH2¼CHCH2O), 99.1,
97.1 (2C, 2C-1), 76.3, 71.6, 69.5, 69.0, 68.8, 68.6, 68.0,
67.5, 63.9, 60.0 (10C, C-2–6, C-20–50, CH2¼CHCH2O).
Anal. calcd for C56H48O16: C 68.84; H 4.95. Found: C
68.58; H 5.15.
J5 a,5 b=12.1 Hz, H-50b), 4.14 (dd, 1H, J5,6a=5.8,
J6a,6b=12.1 Hz, H-6a), 3.43 (dd, 1H, J5,6b=5.0,
J6a,6b=12.1 Hz, H-6b). Anal. calcd for C55H44Cl3NO16:
C 61.09; H 4.10. Found: C 61.18; H 4.33.
0
0
Allyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,
6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-4,6-O-ben-
zylidene-ꢁ-D-mannopyranoside (6). To a cooled solution
(À20 ꢀC) of 5 (1.54 g, 5 mmol) and 4 (5.94 g, 5.5 mmol)
in anhydrous CH2Cl2 (50 mL) was added TMSOTf (18
mL, 0.05 mmol). The mixture was stirred at this tem-
perature for 2 h, and then quenched with Et3N (two
drops). The solvents were evaporated in vacuo to give a
residue, which was purified by silica gel column chro-
matography (2:1 petroleum ether–EtOAc) to give tri-
saccharide 6 as a syrup (4.92 g, 80.3%). [a]D À70.4ꢀ (c
1
0.5, CHCl3); H NMR (400 MHz, CDCl3) d 7.98–7.25
(m, 35H, 7Ph), 5.87 (m, 1H, CH2¼CHCH2O), 5.81 (dd,
1H, J3,4=J4,5=9.9 Hz, H-40), 5.64 (dd, 1H, J2,3=3.2,
J3,4=9.9 Hz, H-30), 5.62 (s, 1H, PhCHO2), 5.45 (dd, 1H,
J2,3=J3,4=6.9 Hz, H-300), 5.32 (dd, 1H, J1,2=5.2,
J2,3=6.9 Hz, H-200), 5.30 (d, 1H, J1,2=1.2 Hz, H-10),
5.30–5.20 (m, 2H, CH2¼CHCH2O), 5.06 (m, 1H, H-400),
4.87 (d, 1H, J1,2=1.1 Hz, H-1), 4.47 (dd, 1H, J1,2=1.2,
J2,3=3.2 Hz, H-20), 4.41 (d, 1H, J1,2=5.2 Hz, H-100),
4.36–4.28 (m, 3H), 4.18–4.09 (m, 5H), 4.01–3.85 (m,
4H), 2.91 (dd, 1H, J5,6b=6.9, J6a,6b=12.1 Hz, H-6b);
13C NMR (100 MHz, CDCl3) d 166.3, 166.2, 165.5,
165.4, 165.3, 164.8 (6C, 6PhCO), 133.7–126.2 (PhCO,
CH2¼CHCH2O), 118.1 (CH2¼CHCH2O), 102.3
(PhCHO2), 99.4, 99.1, 98.8 (3C, 3C-1), 78.8, 75.5, 74.7,
2,3,4-Tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,6-tri-
O-benzoyl-ꢁ-D-mannopyranosyl trichloroacetimidate (4).

4032
J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
71.4, 70.8, 70.3, 70.2, 69.2, 69.0, 69.0, 68.2, 67.3, 63.7,
63.6, 60.2 (15C, C-2–6,
C-20—60, C-200–500,
166.2, 165.8, 165.5, 165.5, 165.4, 165.4, 165.3, 164.9,
164.8 (9C, 9PhCO), 118.1 (CH2¼CHCH2O), 101.7
(PhCHO2), 98.7, 98.1, 97.1, 97.0 (4C, 4C-1), 78.7, 76.6,
74.2, 74.1, 69.9, 69.9, 69.6, 69.5, 68.7, 68.6, 68.4, 68.4,
67.9, 67.7, 67.6, 64.1, 63.9, 60.0, 59.9. Anal. calcd for
C95H82O28: C 68.25; H 4.94. Found: C 68.16; H 5.10.
CH2¼CHCH2O). Anal. calcd for C69H62O21: C 67.53;
H 5.09. Found: C 67.70; H 5.00.
Allyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,
6-tri-O-benzoyl-ꢁ-D-mannopyranosyl- (1!3)-2-O-acetyl-
4,6-O-benzylidene-ꢁ-D-mannopyranoside (7). To a solu-
tion of 6 (123 mg, 0.1 mmol) in pyridine (5 mL) was
added acetic anhydride (2.0 mL, 2 mmol). The reaction
mixture was stirred at rt for 12 h, at the end of which
time TLC (3:1 petroleum ether–EtOAc) indicated that
the reaction was complete. The reaction mixture was
concentrated, and then the residue was purified by flash
column chromatography on a silica gel column (3:1
petroleum ether–EtOAc) to give compound 7 (115 mg,
90.6%) as a foamy solid. [a]D À70.8ꢀ (c 0.5, CHCl3); 1H
NMR (400 MHz, CDCl3) d 8.13–7.24 (m, 35H, 7Ph),
5.87 (dd, 1H, J3,4=J4,5=9.8 Hz, H-40), 5.82 (m, 1H,
CH2¼CHCH2O), 5.64 (s, 1H, PhCHO2), 5.50 (dd, 1H,
J2,3=3.3, J3,4=9.8 Hz, H-30), 5.46 (dd, 1H,
J2,3=J3,4=6.3 Hz, H-300), 5.40 (dd, 1H, J1,2=0.6,
J2,3=3.0 Hz, H-2), 5.28 (d, 1H, J1,2=1.4 Hz, H-10), 5.26
(dd, 1H, J1,2=4.6, J2,3=6.3 Hz, H-200), 5.31–5.21 (m,
2H, CH2¼CHCH2O), 5.01 (m, 1H, H-400), 4.82 (d, 1H,
J1,2=0.6 Hz, H-1), 4.47 (d, 1H, J1,2=4.6 Hz, H-100),
4.43 (dd, 1H, J1,2=1.4, J2,3=3.3 Hz, H-20), 4.37–4.29
(m, 3H), 4.18–4.11 (m, 2H), 4.09 (dd, 1H,
J3,4=J4,5=9.4 Hz, H-4), 4.03–3.84 (m, 4H), 2.99 (dd,
1H, J5,6b=6.2, J6a,6b=12.0 Hz, H-6b). Anal. calcd for
C71H64O22: C 67.18; H 5.08. Found: C 67.37; H 5.25.
Allyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,
6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-tri-O-
benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-benzoyl-ꢁ-
D-mannopyranoside (9). Compound 8 (3.00 g, 1.8
mmol) was dissolved in 50 mL 90% TFA and stirred for
2 h, at the end of which time the reaction mixture was
poured into 200 mL toluene and then the mixture was
concentrated to give a yellowsyrup. Dried under high
vacuum for 2 h, the solid was was dissolved in pyridine
(20 mL), and benzoyl chloride (3.5 mL, 30 mmol) was
added dropwise in 30 min. The mixture was stirred
overnight at room temperature, and TLC (2:1 petro-
leum ether–EtOAc) indicated that the reaction was
complete. Ice water was added, and the mixture was
diluted with dichloromethane, washed with M HCl,
water, and satd aq sodium bicarbonate subsequently.
The organic layer was combined, dried, and con-
centrated. Purification by column chromatography (2:1
petroleum ether–EtOAc) gave 9 (2.96 g, 91.9% for two
steps) as a foamy solid. [a]D À92.9ꢀ (c 1.2, CHCl3); H
1
NMR (400 MHz, CDCl3) d 8.16–7.31 (m, 55H, 11Ph),
5.85 (m, 1H, CH2¼CHCH2O), 5.84 (dd, 1H,
J3,4=J4,5=9.9 Hz, H-4, Manp), 5.76–5.62 (m, 3H), 5.49
(m, 1H, H-4, Xylp), 5.45 (dd, 1H, J1,2=J2,3=5.7 Hz, H-
2, Xylp), 5.35–5.19 (m, 3H), 5.14 (d, 1H, J1,2=0.7 Hz,
H-1, Manp), 5.12 (d, 1H, J1,2=4.2 Hz, H-1, Xylp), 5.06
(m, 1H, H-4, Xylp), 5.01 (dd, 1H, J2,3=5.4, J3,4=6.0
Hz, H-3, Xylp), 4.87 (d, 1H, J1,2=0.9 Hz, H-1, Manp),
4.43 (d, 1H, J1,2=5.1 Hz, H-1, Xylp), 4.36–3.86 (m,
13H), 2.92 (dd, 1H, J=6.1, 12.2 Hz, H-6b, Manp), 2.81
(dd, 1H, J=6.3, 12.1 Hz, H-6b, Manp); 13C NMR
(100 MHz, CDCl3) 166.2, 166.1, 165.6, 165.6, 165.5,
165.4, 165.3, 165.3, 165.1, 164.8, 164.7 (11C, 11PhCO),
118.3 (CH2¼CHCH2O), 100.1, 99.4, 99.0, 96.8 (4C, 4C-
1), 78.2, 76.0, 75.9, 70.3, 69.8, 69.7, 69.3, 68.8, 68.7,
68.6, 68.1, 67.6, 64.0, 63.7, 60.6, 60.5 (some signals
overlapped). Anal. calcd for C102H86O30: C 68.37; H
4.84. Found: C 68.52; H 4.99.
Allyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,6
-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-tri-O-
benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-O-benzylidene-ꢁ-
D-mannopyranoside (8). To a cooled solution (À10 ꢀC)
of 6 (3.68 g, 3.0 mmol) and 2 (2.00 g, 3.3 mmol) in
anhydrous CH2Cl2 (50 mL) was added TMSOTf (54
mL, 0.3 mmol). After stirring at this temperature for 30
min, further TMSOTf (360 mL, 2.0 mmol) and 2.2 equiv
of donor 2 (4.00 g, 6.6 mmol) was added to the reaction
mixture subsequently. The mixture was stirred for 2 h,
and then quenched with Et3N (four drops). The solvents
were evaporated in vacuo to give a residue, which was
purified by silica gel column chromatography (2:1 pet-
roleum ether–EtOAc) to give tetrasaccharide 8 (3.21 g,
64.2%) as a foamy solid. [a]D À67.5ꢀ (c 1.3, CHCl3); 1H
NMR (400 MHz, CDCl3) d 8.29–7.19 (m, 50H, 10Ph),
5.92 (dd, 1H, J2,3=3.2, J3,4=9.8 Hz, H-3, Manp), 5.87
(dd, 1H, J3,4=J4,5=9.8 Hz, H-4, Manp), 5.78 (m, 1H,
CH2¼CHCH2O), 5.66 (dd, 1H, J1,2=J2,3=4.2 Hz, H-2,
Xylp), 5.53 (s, 1H, PhCHO2), 5.52 (dd, 1H, J2,3=5.6,
J3,4=6.0 Hz, H-3, Xylp), 5.40–5.38 (m, 2H, H-2 and
H-4, Xylp), 5.38 (d, 1H, J1,2=1.3, H-1, Manp), 5.33 (dd,
1H, J2,3=4.4, J3,4=6.0 Hz, H-3, Xylp), 5.24–5.12 (m,
3H), 5.08 (m, 1H, H-4, Xylp), 5.04 (d, 1H, J1,2=1.7 Hz,
H-1, Manp), 5.00 (d, 1H, J1,2=4.2 Hz, H-1, Xylp), 4.83
(d, 1H, J1,2=0.7 Hz, H-1, Manp), 4.60 (d, 1H, J1,2=4.4
Hz, H-1, Xylp), 4.52–4.40 (m, 3H), 4.28 (dd, 1H,
J2,3=3.3, J3,4=9.8 Hz, H-3, Manp), 4.20–4.05 (m, 6H),
3.92–3.86 (m, 2H), 3.76 (m, 1H), 3.43 (dd, 1H,
J3,4=J4,5=9.8 Hz, H-4, Manp), 3.02 (dd, 1H, J=6.3,
12.0 Hz, H-6b, Manp); 13C NMR (100 MHz, CDCl3) d
2,3,4-Tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-3,4,6-tri-
O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2, 3,4-tri-O-ben-
zoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-benzoyl-ꢁ-D-
mannopyranosyl trichloroacetimidate (10). To a solution
of 9 (2.69 g, 1.5 mmol) in 90% acetic acid (15 mL)
containing sodium acetate (0.44 g, 4.5 mmol) was added
PdCl2 (81 mg, 0.75 mmol), and the mixture was stirred
for 12 h, at the end of which time TLC (2:1 petroleum
ether–EtOAc) indicated that the reaction was complete.
The mixture was diluted with dichloromethane (150
mL), washed with water and satd aq sodium bicarbo-
nate. The organic layer was concentrated, and the resi-
due was passed through a short silica gel column with
1:1 petroleum ether–EtOAc as the eluent to give crude
2,3,4-tri-O-benzoyl-b-d-xylopyranosyl-(1!2)-3,4,6-tri-O-
benzoyl-a-d-mannopyranosyl-(1!3)[2,3,4-tri-O-benzoyl-
b-d-xylopyranosyl-(1!2)]-4,6-di-O-benzoyl-d-manno-

J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
4033
pyranose as a syrup. Dried under high vacuum for 2 h,
the solid was was dissolved in dichloromethane (10 mL),
and CCl3CN (0.5 mL, 5 mmol) and DBU (40 mL, 0.3
mmol) were added. The reaction mixture was stirred for
2 h, at the end of which time TLC (2:1 petroleum ether–
EtOAc) indicated that the reaction was complete. Con-
centration of the reaction mixture, followed by pur-
ification of the crude product on a silica gel column with
2:1 petroleum ether–EtOAc as the eluent, furnished the
tetrasaccharide donor 10 (2.44 g, 85.8%) as a foamy
164.5 (11C, 11PhCO), 99.8, 99.5, 98.7, 98.4, 98.2 (5C,
5C-1), 75.6, 71.9, 71.3, 70.0, 69.7, 69.4, 69.3, 68.5, 68.2,
67.8, 67.5, 64.7, 63.3, 61.6, 60.2, 59.9 (C-2–6, Manp;
C-2–5, Xylp, some signals overlapped), 54.4 (OCH3),
24.4 (CH3CO). Anal. calcd for C108H96O36: C 65.85; H
4.91. Found: C 65.71; H 5.13.
Methyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-
tri-O-benzoyl-ꢁ-D-xylopyranosyl-(1!2)-]4,6-di-O-ben-
zoyl-ꢁ-D-mannopyranosyl-(1!3)-2-O-acetyl-4,6-di-O-
benzoyl-ꢁ-D-mannopyranoside (14). To a solution of 13
(1.00 g, 0.51 mmol) in pyridine (10 mL) was added
benzoyl chloride (1.2 mL, 10 mmol) and DMAP (12 mg,
0.1 mmol). The reaction mixture was stirred at 70 ꢀC for
12 h, at the end of which time TLC (1:1 petroleum
ether–EtOAc) indicated that the reaction was complete,
and then quenched with MeOH (2.0 mL). The reaction
mixture was concentrated, and purification of the resi-
due by column chromatography on a silica gel column
(2:1 petroleum ether–EtOAc) gave compound 14 (877
solid. [aD À71.2ꢀ (c 1.3, CHCl3); H NMR (400 MHz,
1
CDCl3) d 8.70 (s, 1H, CNHCCL3), 8.13–7.31 (m, 55H,
11Ph), 6.40 (s, 1H, H-1, Manp), 5.82 (dd, 1H,
J3,4=J4,5=9.7 Hz, H-4, Manp), 5.78 (dd, 1H, J2,3=3.2,
J3,4=9.8 Hz, H-3, Manp), 5.74 (dd, 1H, J1,2=J2,3=4.1
Hz, H-2, Xylp), 5.72 (dd, 1H, J3,4=J4,5=9.8 Hz, H-4,
Manp), 5.51 (m, 1H, H-4, Xylp), 5.45 (dd, 1H,
J1,2=J2,3=4.0 Hz, H-2, Xylp), 5.36 (dd, 1H, J2,3=4.0,
J3,4=6.5 Hz, H-3, Xylp), 5.16 (d, 1H, J1,2=4.1 Hz, H-1,
Xylp), 5.06 (d, 1H, J1,2=1.7 Hz, H-1, Manp), 5.04 (m,
1H, H-4, Xylp), 4.58 (d, 1H, J1,2=4.0 Hz, H-1, Xylp).
Anal. calcd for C101H82Cl3NO30: C 63.98; H 4.36.
Found: C 63.85; H 4.51.
mg, 79.3%) as a syrup. [a]D À48.8ꢀ (c 0.5, CHCl3); H
1
NMR (400 MHz, CDCl3) d 8.02–7.16 (m, 65H, 13Ph),
5.90 (dd, 1H, J3,4=J4,5=9.9 Hz, H-4, Manp), 5.71 (dd,
1H, J2,3=3.2, J3,4=9.8 Hz, H-3, Manp), 5.69 (dd, 1H,
J3,4=J4,5=9.8 Hz, H-4, Manp), 5.64 (dd, 1H,
J3,4=J4,5=9.6 Hz, H-4, Manp), 5.38–5.34 (m, 3H), 5.32
(dd, 1H, J1,2=1.0, J2,3=3.1 Hz, H-2, Manp), 5.23 (dd,
1H, J1,2=J2,3=5.1 Hz, H-2, Xylp), 5.18 (dd, 1H,
J2,3=5.1, J3,4=6.3 Hz, H-3, Xylp), 5.06 (d, 1H, J1,2=0.7
Hz, H-1, Manp), 5.01 (d, 1H, J1,2=0.8 Hz, H-1, Manp),
4.98 (m, 1H, H-4, Xylp), 4.70 (d, 1H, J1,2=1.0 Hz, H-1,
Manp), 4.55 (d, 1H, J1,2=4.9 Hz, H-1, Xylp), 4.50 (d,
1H, J1,2=5.1 Hz, H-1, Xylp), 4.40–4.28 (m, 10H),
4.07–3.84 (m, 6H), 3.34 (s, 3H, OCH3), 2.90 (dd, 1H,
J=6.0, 11.9 Hz, H-6, Manp), 2.04 (s, 3H, CH3CO); 13C
NMR (100 MHz, CDCl3) d 170.3 (CH3CO), 166.0,
165.9, 165.8, 165.4, 165.3, 165.3, 165.1, 165.1, 164.9,
164.8, 164.6, 164.5, 164.4 (13C, 13PhCO), 99.8, 98.6,
98.6, 98.4, 98.4 (5C, 5C-1), 76.4, 74.7, 74.2, 73.6, 70.9,
69.7, 69.6, 69.3, 69.1, 68.8, 68.4, 68.3, 68.2, 67.8, 63.6,
63.3, 63.2, 60.4, 60.3, 60.3 (C-2–6, Manp; C-2–5, Xylp,
some signals overlapped), 55.1 (OCH3), 22.6 (CH3CO).
Anal. calcd for C122H104O38: C 67.27; H 4.81. Found: C
67.08; H 4.77.
Methyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-
tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-ben-
zoyl-ꢁ-D-mannopyranosyl-(1!3)-2-O-acetyl-ꢁ-D-manno-
pyranoside (13). To a cooled solution (À20 ꢀC) of 10
(1.90 g, 1.0 mmol) and 11 (0.42 g, 1.5 mmol) in anhy-
drous CH2Cl2 (50 mL) was added TMSOTf (18 mL, 0.1
mmol). The mixture was stirred at this temperature for
2 h, and then quenched with Et3N (two drops). The
solvents were evaporated in vacuo to afford the crude
pentasaccharide 12. Compound 12 was dissolved in
pyridine (20 mL), and Ac2O (10 mL) was added. The
mixture was stirred at rt for 12 h, and then was evapo-
rated and coevaporated with toluene in vacuo to give a
residue. The residue was dissolved in 50 mL 90% TFA
and stirred for 2 h, at the end of which time the reaction
mixture was poured into 200 mL toluene and then the
mixture was concentrated. The residue was purified by
silica gel column chromatography (1:1 petroleum ether–
EtOAc) to give 13 (1.07 g, 54.3% for three steps) as a
syrup. [a]D À41.3ꢀ (c 1.3, CHCl3); H NMR (400 MHz,
1
CDCl3) d 8.07–6.86 (m, 55H, 11Ph), 5.76 (dd, 1H,
J3,4=J4,5=9.8 Hz, H-4, Manp), 5.74 (dd, 1H, J2,3=3.1,
J3,4=9.8 Hz, H-3, Manp), 5.66 (dd, 1H, J3,4=J4,5=9.8
Hz, H-4, Manp), 5.58 (dd, 1H, J1,2=J2,3=4.6 Hz, H-2,
Xylp), 5.39–5.36 (m, 2H), 5.31 (d, 1H, J1,2=1.0 Hz,
H-1, Manp), 5.29 (m, 1H, H-4, Xylp), 5.24 (dd, 1H,
J1,2=1.4, J2,3=3.2 Hz, H-2, Manp), 5.20 (dd, 1H,
J1,2=J2,3=5.5 Hz, H-2, Xylp), 5.06 (m, 1H, H-4, Xylp),
5.02 (d, 1H, J1,2=0.6 Hz, H-1, Manp), 4.93 (d, 1H,
J1,2=0.8 Hz, H-1, Manp), 4.63 (d, 1H, J1,2=4.4 Hz,
H-1, Xylp), 4.57 (d, 1H, J1,2=5.5 Hz, H-1, Xylp),
4.42–4.34 (m, 4H), 4.29–4.27 (m, 2H), 4.17 (dd, 1H,
J2,3=3.2, J3,4=9.9 Hz, H-3, Manp), 4.10–4.05 (m, 3H),
4.00–3.93 (m, 2H), 3.87–3.81 (m, 3H), 3.69–3.57 (m,
2H), 3.29 (s, 3H, OCH3), 2.89 (dd, 1H, J=6.2 Hz, 12.2,
H-6b, Manp), 2.04 (s, 3H, CH3CO); 13C NMR
(100 MHz, CDCl3) d 170.1 (CH3CO), 167.3, 166.0,
165.5, 165.3, 165.2, 165.1, 165.1, 164.9, 164.8, 164.6,
Methyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-
tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-ben-
zoyl-ꢁ-D-mannopyranosyl-(1!3)-4,6-di-O-benzoyl-ꢁ-D-
mannopyranoside (15). To a solution of 14 (762 mg,
0.35 mmol) in anhydrous CH2Cl2 (10 mL) was added
anhyd MeOH (50 mL), then acetyl chloride (1.5 mL)
was added to the reaction mixture at 0 ꢀC. The solution
was stoppered in a flask and stirred at room tempera-
ture until TLC (3:1 petroleum ether–EtOAc) showed
that the starting material disappeared. The solution was
neutralized with Et3N, then concentrated to dryness.
The residue was passed through a short silica gel col-
umn to give 15 (664 mg, 88.9%) as a foamy solid. [a]D
À27.4ꢀ (c 1.3, CHCl3); H NMR (400 MHz, CDCl3) d
1
8.12–6.99 (m, 65H, 13Ph), 5.83 (dd, 1H, J3,4=J4,5=10.0
Hz, H-4, Manp), 5.72 (dd, 1H, J3,4=J4,5=9.5 Hz, H-4,

4034
J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
Manp), 5.65 (dd, 1H, J2,3=3.3, J3,4=10.0 Hz, H-3,
Manp), 5.57 (dd, 1H, J3,4=J4,5=9.8 Hz, H-4, Manp),
5.51 (dd, 1H, J1,2=J2,3=5.1 Hz, H-2, Xylp), 5.41–5.35
(m, 4H), 5.29 (dd, 1H, J1,2=J2,3=4.5 Hz, H-2, Xylp),
5.20 (dd, 1H, J2,3=5.1, J3,4=5.3 Hz, H-3, Xylp), 5.07
(d, 1H, J1,2=1.1 Hz, H-1, Manp), 5.03 (d, 1H, J1,2=0.6
Hz, H-1, Manp), 5.02 (d, 1H, J1,2=4.5 Hz, H-1, Xylp),
5.01 (m, 1H, H-4, Xylp), 4.58 (d, 1H, J1,2=0.9, H-1,
Manp), 4.54 (dd, 1H, J2,3=2.9, J3,4=9.9 Hz, H-3,
Manp), 4.49 (d, 1H, J1,2=5.1 Hz, H-1, Xylp), 4.42–4.26
(m, 9H), 4.11–3.86 (m, 6H), 3.36 (s, 3H, OCH3); 13C
NMR (100 MHz, CDCl3) d 166.1, 166.1, 165.7, 165.4,
165.4, 165.2, 165.1, 165.1, 165.0, 164.9, 164.8, 164.7,
164.7 (13C, 13PhCO), 100.7, 99.9, 99.5, 98.8, 98.7 (5C,
5C-1), 77.7, 75.0, 74.9, 70.2, 69.9, 69.7, 69.6, 69.5, 68.6,
68.3, 68.2, 68.1, 68.0, 67.9, 64.2, 63.4, 63.3, 60.2, 60.1
(C-2–6, Manp; C-2–5, Xylp, some signals overlapped),
55.0 (OCH3). Anal. calcd for C120H102O37: C 67.47; H
4.81. Found: C 67.72; H 5.01.
MeONa–MeOH (0.2 N) was added to the solvent sub-
sequently. After 96 h at room temperature, water (18
mL, 1.0 mmol) was added to the mixture to cleave the
methyl ester. After stirring at room temperature for 2 h,
the reaction mixture was concentrated and purified on a
Bio-Gel P2 column (eluent: water), affording the target
hexasaccharide 18 (63 mg, 64.5%) as a foamy solid. [a]D
+56.9ꢀ (c 0.5, H2O); H NMR (D2O, 400 MHz): d 5.05
1
(s, 1H, H-1, Manp), 4.97 (s, 1H, H-1, Manp), 4.65 (s,
1H, H-1, Manp), 4.49 (d, 1H, J1,2=8.0 Hz, H-1,
GluAp), 4.28 (d, 1H, J1,2=8.8 Hz, H-1, Xylp), 4.24 (d,
1H, J1,2=8.4 Hz, H-1, Xylp), 3.28 (s, 3H, OCH3); 13C
NMR (100 MHz, D2O): 173.9 (–COONa), 103.5 (C-1,
JC1, H1=163.4 Hz, Xylp), 103.5 (C-1, JC1, H1=163.4 Hz,
Xylp), 102.7 (C-1, JC1, H1=176.2 Hz, Manp), 100.6
(C-1, JC1,H1=172.7 Hz, Manp), 99.1 (C-1,
JC1,H1=174.1 Hz, Manp), 96.0 (C-1, JC1, H1=166.8 Hz,
GluAp), 78.4, 78.2, 76.2, 76.1, 76.0, 75.7, 74.1, 73.5,
73.4, 72.8, 72.2, 70.5, 70.2, 69.4, 66.9, 66.5, 66.2, 65.3,
65.3, 61.5, 60.4, 60.4 (C-2–6, Manp; C-2–5, Xylp; C-2–5,
GluAp; some signals overlapped), 55.0 (O–CH3). Nega-
tive-ESI-MS calcd for C35H57O30Na: [M] 980.8, Found:
[MÀNa] 957.9.
Methyl 2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-
tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-ben-
zoyl-ꢁ-D-mannopyranosyl-(1!3)-[methyl 2,3,4-tri-O-ace-
tyl-ꢀ-D-glucopyranosyluronate-(1!2)-]4,6-di-O-benzoyl-
ꢀ-D-mannopyranoside (17). To a cooled solution (0 ꢀC)
of 15 (427 mg, 0.2 mmol) and methyl 2,3,4-tri-O-acetyl-
a-d-glucopyranosyluronate trichloroacetimidate 16 (144
mg, 0.3 mmol) in anhydrous CH2Cl2 (10 mL) was added
TMSOTf (8 mL, 0.05 mmol). The mixture was stirred at
this temperature for 2 h, and then quenched with Et3N
(one drop). The solvents were evaporated in vacuo to
give a residue, which was purified by silica gel column
chromatography (1:1 petroleum ether–EtOAc) to give
17 (356 mg, 72.7%) as a foamy solid. [a]D À61.7ꢀ (c 1.3,
2,3-Di-O-acetyl-4,6-O-isopropylidene-ꢁ-D-mannopyrano-
syl trichloroacetimidate (19). Compound 1,2,3-O-acetyl-
4,6-O-isopropylidene-d-mannopyranoside (6.9 g, 20.0
mmol) was dissolved in 1 M solution of ammonia–
methanol (100 mL) and stirred for 4 h, at the end of
which time TLC (3:1 petroleum ether–EtOAc) indicated
that the reaction was complete. The solution was con-
centrated and dried under high vacuum giving a white
foamy solid. This foamy solid was dissolved in dry di-
chloromethane (50 mL), then trichloroacetonitrile (6.3
mL, 30 mmol) and 1,8-diazabicyclo[5.4.0]undecene
(DBU) (0.50 mL, 4.04 mmol) was added subsequently.
The reaction mixture was stirred under nitrogen for 3 h
and then concentrated. The residue was purified by
chromatography (4:1 petroleum ether–EtOAc) to give
19 (7.40 g, 82.2%) as a syrup. [a]D +42ꢀ (c 1.0, CHCl3);
1H NMR (400 MHz, CDCl3) d 8.72 (s, 1H, CNHCCL3),
6.17 (s, 1H, J1,2=1.6 Hz, H-1), 5.50 (dd, 1H, J1,2=1.6,
J2,3=3.4 Hz, H-2), 5.30 (dd, 1H, J2,3=3.4, J3,4=10.3
Hz, H-3), 4.11 (dd, 1H, J3,4=10.3, J4,5=10.0 Hz, H-4),
3.98–3.85 (m, 3H), 2.20 (s, 3H, CH3CO), 2.04 (s, 3H,
CH3CO), 1.55 (s, 3H, isopropylidene), 1.42 (s, 3H, iso-
propylidene). Anal. calcd for C15H20Cl3NO8: C 40.12;
H 4.49. Found: C 40.40; H 4.73.
1
CHCl3); H NMR (400 MHz, CDCl3) d 8.20–6.90 (m,
65H, 13 PhH), 6.55 (dd, 1H, J1,2=J2,3=7.7 Hz, H-2,
Xylp), 6.09 (dd, 1H, J3,4=J4,5=9.6 Hz, H-4, Manp),
6.01 (dd, 1H, J3,4=J4,5=8.8 Hz, H-4, Manp), 5.78 (dd,
1H, J3,4=J4,5=9.9 Hz, H-4, Manp), 5.28 (d, 1H,
J1,2=1.1 Hz, H-1, Manp), 5.18 (d, 1H, J1,2=0.8 Hz,
H-1, Manp), 5.12 (d, 1H, J1,2=1.1 Hz, H-1, Manp), 4.81
(d, 1H, J1,2=7.9 Hz, H-1, Xylp), 4.77 (d, 1H, J1,2=7.7
Hz, H-1, Xylp), 4.39 (dd, 1H, J1,2=1.1, J2,3=3.2 Hz,
H-2, Manp), 4.17 (dd, 1H, J1,2=0.8, J2,3=2.8 Hz, H-2,
Manp), 4.07 (d, 1H, J1,2=7.2 Hz, H-1, GluAp), 3.79
(dd, 1H, J1,2=1.1, J2,3=2.9 Hz, H-2, Manp), 3.52 (s,
3H, COOCH3), 3.18 (s, 3H, OCH3), 2.15, 2.13, 2.01 (3s,
9H, 3COCH3). 13C NMR (100 MHz, CDCI3): 171.3,
171.0, 169.7, 169.2 (4C, 3COCH3, COOMe), 167.3.
167.2, 166.1, 166.0, 165.8, 165.4, 165.3, 165.3, 1653,
165.2, 165.2, 165.1, 164.6 (13C, 13COPh), 102.6, 100.6,
100.6, 99.6, 98.9, 98.2 (6C, 6C-1), 54.7 (OCH3), 52.7
(COOCH3), 208, 20.7, 20.5 (3C, 3COCH3). Anal. calcd
for C133H118O46: C, 65.13; H, 4.85. Found: C, 65.32; H,
4.97.
Methyl 2,3-di-O-acetyl-4,6-O-isopropylidene-ꢁ-D-manno-
pyranosyl-(1!3)-4,6-O-isopropylidene-ꢁ-D-mannopyra-
noside (21). To a cooled solution (À20 ꢀC) of 20 (1.17
g, 5 mmol) and 19 (2.46 g, 5.5 mmol) in anhyd CH2Cl2
(50 mL) was added TMSOTf (18 mL, 0.05 mmol). The
mixture was stirred at this temperature for 2 h, and then
quenched with Et3N (two drops). The solvents were
evaporated in vacuo to give a residue, which was pur-
ified by silica gel column chromatography (2:1 petro-
leum ether–EtOAc) to give disaccharide 21 (1.93 g,
74.2%) as a syrup. [a]D +43.3ꢀ (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3) d 5.36 (dd, 1H, J1,2=1.0,
J2,3=3.2 Hz, H-20), 5.21 (dd, 1H, J2,3=3.2, J3,4=9.9
Hz, H-30), 5.20 (s, 1H, J1,2=1.0 Hz, H-10), 4.73 (s, 1H,
Methyl ꢀ-D-xylopyranosyl-(1!2)-ꢁ-D-mannopyranosyl-
(1!3)-[ꢀ-D-xylopyranosyl-(1!2)-]ꢁ-D-mannopyranosyl-
(1!3)-[(ꢀ-D-glucopyranosyluronic
acid)-(1!2)-]ꢁ-D-
mannopyranoside, sodium salt (18). Hexasaccharide 17
(245 mg, 0.10 mmol) was dissolved in anhydrous
CH2Cl2 (5 mL), then MeOH (30 mL) and 1 mL of

J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
4035
J1,2=1.1 Hz, H-1), 4.14 (dd, 1H, J3,4=J4,5=9.9 Hz,
H-4), 4.06–4.02 (m, 2H), 3.95 (dd, 1H, J2,3=3.3,
J3,4=10.0 Hz, H-3), 3.87–3.76 (m, 5H), 3.60 (m, 1H,
H-5), 3.36 (s, 3H, OCH3), 2.17 (s, 3H, CH3CO), 2.15 (s,
3H, CH3CO), 1.52 (s, 6H, isopropylidene), 1.40 (s, 3H,
isopropylidene), 1.29 (s, 3H, isopropylidene); 13C NMR
(100 MHz, CDCI3): 170.0, 169.6 (2C, 2COCH3), 101.4
(1C, Me2CO2), 100.1, 100.0 (2C, 2C-1), 99.5 (1C,
Me2CO2), 74.1, 71.6, 71.1, 70.0, 68.9, 68.6, 65.4, 64.2,
62.4, 62.2 (10C, C2–6, C20–60), 54.9 (OCH3), 29.1, 29.0,
20.8, 20.7 [4C, 2(CH3)2CO2], 19.3, 19.1 (2C, 2COCH3).
Anal. calcd for C23H36O13: C, 53.07; H, 6.97. Found: C,
53.32; H, 6.70.
stirred at this temperature for 2 h, and then quenched
with Et3N (two drops). The solvents were evaporated in
vacuo to give a residue, which was purified by silica gel
column chromatography (1:1 petroleum ether–EtOAc)
to give trisaccharide 25 (3.20 g, 67.2%) as a syrup. [a]D
ꢀ
1
+91.7 (c 1.0, CHCl3); H NMR (CDCl3, 400 MHz): d
7.95–7.34 (m, 15H,
3
PhH), 5.94 (dd, 1H,
J3,4=J4,5=10.0 Hz, H-4), 5.75 (dd, 1H, J2,3=3.3 Hz,
J3,4=10.0 Hz, H-3), 5.53 (dd, 1H, J1,2=1.8 Hz,
J2,3=3.3 Hz, H-2), 5.43 (d, 1H, J1,2=1.8 Hz, H-1), 5.31
(d, 1H, J1,2=1.0 Hz, H-1), 4.71 (d, 1H, J2,1=1.0 Hz, H-
1), 4.66–4.58 (m, 3H), 4.26–4.18 (m, 4H), 4.04–3.99 (m,
2H), 3.90–3.82 (m, 4H), 3.68–3.63 (m, 2H), 3.36 (s, 3H,
OCH3), 2.17 (s, 3H, COCH3), 1.56, 1.45 (2s, 6H, iso-
propylidene), 1.37 (s, 6H, isopropylidene); 13C NMR
(100 MHz, CDCl3): 169.5 (COCH3), 166.3, 165.6, 163.6
(5C, 5 COPh), 101.2, 100.8, (2C, 2 Me2C) 99.9, 99.4,
98.3 (3C, 3 C-1), 54.8 (OCH3), 29.1, 28.9 (2C,
CH3CCH3), 20.6 (COCH3), 19.2, 19.0 (2C, CH3CCH3).
Anal. calcd for C48H56O20: C, 60.50; H, 5.92. Found: C,
60.45; H, 5.66.
Methyl 2,3-di-O-acetyl-4,6-O-isopropylidene-ꢁ-D-manno-
pyranosyl-(1!3)-2-O-acetyl-4,6-O-isopropylidene-ꢁ-D-
mannopyranoside (22). To a solution of 21 (104 mg, 0.2
mmol) in pyridine (5 mL) was added acetic anhydride
(2.0 mL, 2 mmol). The reaction mixture was stirred at rt
for 12 h, at the end of which time TLC (3:1 petroleum
ether–EtOAc) indicated that the reaction was complete.
The reaction mixture was concentrated, and then the
residue was purified by flash column chromatography
on a silica gel column (3:1 petroleum ether–EtOAc) to
give compound 22 (100 mg, 89.3%) as a foamy solid.
[a]D +38.9ꢀ (c 1.5, CHCl3); 1H NMR (400 MHz,
CDCl3) d 5.37 (dd, 1H, J1,2=1.2, J2,3=3.3 Hz, H-20),
5.34 (dd, 1H, J1,2=1.5, J2,3=3.0 Hz, H-2), 5.26 (dd, 1H,
J2,3=3.3, J3,4=9.8 Hz, H-30), 5.12 (s, 1H, J1,2=1.2 Hz,
H-10), 4.70 (s, 1H, J1,2=1.5 Hz, H-1), 4.20 (dd, 1H,
J3,4=J4,5=9.9 Hz, H-4), 4.11–3.87 (m, 8H), 3.66 (m,
1H, H-5), 3.34 (s, 3H, OCH3), 2.21 (s, 3H, CH3CO),
2.19 (s, 3H, CH3CO), 2.14 (s, 3H, CH3CO), 1.55 (s, 3H,
isopropylidene), 1.53 (s, 3H, isopropylidene),1.38 (s,
3H, isopropylidene), 1.31 (s, 3H, isopropylidene). Anal.
calcd for C25H38O14: C, 53.37; H, 6.81. Found: C, 53.41;
H, 6.62.
Methyl 2-O-acetyl-3,4,6-tri-O-benzoyl-ꢁ-D-mannopyra-
nosyl-(1!3)-2-O-acetyl-4,6-O-isopropylidene-ꢁ-D-man-
nopyranosyl-(1!3)-2-O-acetyl-4,6-O-isopropylidene-ꢁ-
D-mannopyranoside (26). To a solution of 25 (95 mg,
0.1 mmol) in pyridine (5 mL) was added acetic anhy-
dride (2.0 mL, 2 mmol). The reaction mixture was stir-
red at rt for 12 h, at the end of which time TLC (3:1
petroleum ether–EtOAc) indicated that the reaction was
complete. The reaction mixture was concentrated, and
then the residue was purified by flash column chroma-
tography on a silica gel column (3:1 petroleum ether–
EtOAc) to give compound 26 (83 mg, 80.6%) as a
foamy solid. [a]D +82.6ꢀ (c 1.0, CHCl3); 1H NMR
(CDCl3, 400 MHz): d 8.10–7.23 (m, 65H, 13 PhH), 6.04
(dd, 1H, J3,4=J4,5=10.0 Hz, H-4), 5.64 (dd, 1H,
J2,3=3.2 Hz, J3,4=10.0 Hz, H-3), 5.45 (dd, 1H,
J1,2=2.0 Hz, J2,3=2.9 Hz, H-2), 5.40 (dd, 1H, J1,2=1.3
Hz, J2,3=3.4 Hz, H-2), 5.32 (d, 1H, J1,2=1.7 Hz, H-1),
5.22 (dd, 1H, J1,2=1.3 Hz, J2,3=2.9 Hz, H-2), 5.10 (d,
1H, J1,2=1.2 Hz, H-1), 4.67 (dd, 1H, J2,3=2.9 Hz,
J3,4=12.1 Hz, H-3), 4.63 (d, 1H, J1,2=1.2 Hz, H-1),
4.46–4.38 (m, 2H), 4.09–4.05 (m, 4H), 3.87–3.81 (m, 4H),
3.70–3.60 (m, 2H), 3.36 (s, 3H, OCH3), 2.31, 2.18, 2.07
(3s, 9H, 3 COCH3), 1.56, 1.52 (2s, 6H, isopropylidene),
1.37 (s, 6H, isopropylidene). Anal. calcd for C52H60O22:
C, 60.22; H, 5.83. Found: C, 60.47; H, 5.61.
Methyl 4,6 - O - isopropylidene - ꢁ - D - mannopyranosyl-
(1!3)-4,6-O-isopropylidene-ꢁ-D-mannopyranoside (23).
Disaccharide 21 (2.60 mg, 5.0 mmol) was dissolved in a
satd methanolic ammonia (25 mL). After 2 h at room
temperature, the reaction mixture was concentrated,
and the residue was purified by by flash column chro-
matography on a silica gel column (EtOAc) to give
compound 23 (2.10 g, 96.3%) as a foamy solid. [a]D
+90.6ꢀ (c 0.5, CHCl3); H NMR (400 MHz, CDCl3) d
1
5.21 (s, 1H, H-10), 4.69 (s, 1H, H-1), 3.34 (s, 3H,
CH3CO), 1.52 (s, 3H, isopropylidene), 1.49 (s, 3H, iso-
propylidene), 1.43 (s, 3H, isopropylidene), 1.37 (s, 3H,
isopropylidene). 13C NMR (100 MHz, CDCI3): 101.4,
101.3 (2C, 2Me2CO2), 100.2, 99.8 (2C, 2C-1), 73.6, 71.4,
71.3, 71.2, 70.9, 68.9, 64.7, 64.3, 62.3, 62.2 (10C, C2–6,
C20–60), 54.9 (OCH3), 29.3, 29.2, 19.4, 19.3 [4C,
2(CH3)2CO2]. Anal. calcd for C19H32O11: C, 52.28; H,
7.39. Found: C, 52.51; H, 7.67.
Methyl 2-O-acetyl-3,4,6-tri-O-benzoyl-ꢁ-D-mannopyra-
nosyl-(1!3)-[2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-
(1!2)-]4,6-di-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-
[2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-
benzoyl-ꢁ-D-mannopyranoside (28). To a cooled solution
(À10 ꢀC) of 25 (1.90 g, 0.2 mmol) and 2 (303 mg, 0.5
mmol) in anhyd CH2Cl2 (30 mL) was added TMSOTf
(18 L, 0.1 mmol). After stirring at this temperature for
30 min, further TMSOTf (72 mL, 0.4 mmol) was added
to the reaction mixture and stirred for 20 min, then
donor 2 (485 mg, 0.8 mmol) was added to the reaction
mixture. The reaction mixture was stirred for 2 h, and
then quenched with Et3N (four drops). The solvents
were evaporated in vacuo to give a residue, which was
Methyl 2-O-acetyl-3,4,6-tri-O-benzoyl-ꢁ-D-mannopyra-
nosyl-(1!3)-4,6-O-isopropylidene-ꢁ-D-mannopyranosyl-
(1!3)-4,6-O-isopropylidene-ꢁ-D-mannopyranoside (25).
To a cooled solution (À20 ꢀC) of 23 (2.18 g, 5 mmol)
and 24 (3.73 g, 5.5 mmol) in anhyd CH2Cl2 (50 mL) was
added TMSOTf (18 mL, 0.05 mmol). The mixture was

4036
J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
dissolved in 50 mL 90% TFA and stirred for 2 h, at the
end of which time the reaction mixture was poured into
200 mL toluene and then the mixture was concentrated.
Dried under high vacuo for 2 h, the residue was dis-
solved in pyridine (10 mL), and benzoyl chloride (1 mL)
was added. The mixture was stirred at rt for 12 h, and
then was evaporated and coevaporated with toluene in
vacuo to give a residue. The residue was purified by
silica gel column chromatography (3:1:1 petroleum
ether–toluene–EtOAc) to give 28 (1.84 g, 42.3% for
O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-benzoyl-
ꢁ-D-mannopyranoside (30). To a cooled solution (0 ꢀC)
of 29 (640 mg, 0.3 mmol) and methyl 2,3,4-tri-O-acetyl-
a-d-glucopyranosyluronate trichloroacetimidate 16 (240
mg, 0.5 mmol) in anhyd CH2Cl2 (10 mL) was added
TMSOTf (8 mL, 0.05 mmol). The mixture was stirred at
this temperature for 2 h, and then quenched with Et3N
(1 drop). The solvents were evaporated in vacuo to give
a residue, which was purified by silica gel column chro-
matography (1:1 petroleum ether–EtOAc) to give hex-
asaccharide 30 (644 mg, 87.6%) as a foamy solid. [a]D
three steps) as a syrup. [a]D À58.6ꢀ (c 1.0, CHCl3); H
1
À22.5ꢀ (c 0.5, CHCl3); H NMR (CDCl3, 400 MHz): d
1
NMR (CDCl3, 400 MHz): d 8.11–7.34 (m, 65H, 13
PhH), 5.94 (dd, 1H, J3,4=J4,5=10.0 Hz, H-4, Manp),
5.77 (dd, 1H, J2,3=3.2 Hz, J3,4=10.0 Hz, H-3, Manp),
5.71 (dd, J1,2=J2,3=6.3 Hz, H-2, Xylp), 5.59 (dd, 1H,
J3,4=J4,5=10.0 Hz, H-4, Manp), 5.54 (dd, 1H,
J3,4=J4,5=9.9 Hz, H-4, Manp), 5.47–5.40 (m, 3H),
5.31–5.27 (m, 2H), 5.18 (d, 1H, J1,2=0.7 Hz, H-1,
Manp), 516 (dd, 1H, J1,2=1.0 Hz, J2,3=3.0 Hz, H-2,
Manp), 4.99 (d, 1H, J1,2=0.8 Hz, H-1, Manp), 4.80 (d,
1H, J1,2=4.8 Hz, H-1, Xylp), 4.53 (d, 1H, J1,2=1.0 Hz,
H-1, Manp), 4.45 (d, 1H, J1,2=6.1 Hz, H-1, Xylp), 3.19
(s, 3H, OCH3), 1.90 (s, 3H, COCH3); 13C NMR
(100 MHz, CDCI3): 168.9 (COCH3), 166.0, 165.9, 165.8,
165.4, 165.3, 165.3, 165.3, 165.2, 165.1, 165.0, 164.9,
164.7, 164.6 (13C, 13 COPh), 99.7, 99.7, 99.7, 99.0, 98.5
(5C, 5 C-1), 54.8 (OCH3), 20.3 (COCH3). Anal. calcd for
C122H104O38: C, 67.27; H, 4.81. Found: C, 67.45; H, 5.96.
6.00 (dd, 1H, J3,4=J4,5=10.0 Hz, H-4, Manp), 5.71 (dd,
1H, J2,3=3.2 Hz, J3,4=10.0 Hz, H-3, Manp), 5.15 (d,
1H, J1,2=1.0 Hz, H-1, Manp), 4.96 (d, 1H, J1,2=0.9 Hz,
H-1, Manp), 4.75 (d, 1H, J1,2=4.9 Hz, H-1, Xylp), 4.60
(d, 1H, J1,2=4.8 Hz, H-1, Xylp), 4.56 (d, 1H, J1,2=1.0
Hz, H-1, Manp), 4.04 (d, 1H, J1,2=6.6 Hz, H-1,
GluAp), 3.69 (s, 3H, COOCH3), 3.23 (s, 3H, OCH3),
1.96, 1.92, 1.31 (3s, 9H, 3 COCH3); 13C NMR
(100 MHz, CDCI3): 167.0, 168.5, 168.5,168.3 (4C, 3
COCH3, COOMe), 165.9, 165.9, 165.9, 165.4, 165.4,
165.4, 165.3, 165.2, 165.1, 165.0, 164.9, 164.6, 164.6
(13C, 13 COPh), 100.9 (C-1, JC1, H1=175 Hz, Manp),
100.3 (C-1, JC1, H1=163 Hz, GluAp), 99.9 (C-1, JC1,
H1=164 Hz, Xylp), 99.5 (C-1, JC1, H1=163 Hz, Xylp),
98.5 (C-1, JC1, H1=172 Hz, Manp), 95.2 (C-1,
JC1,H1=176 Hz, Manp), 54.8 (OCH3), 52.3 (COOCH3),
20.5, 20.3, 20.2 (COCH3). Anal. calcd for C133H118O46:
C, 65.13; H, 4.85. Found: C, 65.45; H, 4.56.
Methyl 3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-
[2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-
benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-tri-O-benzoyl-
ꢀ-D-xylopyranosyl-(1!2)-]4,6-di-O-benzoyl-ꢁ-D-manno-
pyranoside (29). To a solution of 28 (1.09 g, 0.05 mmol)
in anhyd CH2Cl2 (10 mL) was added anhyd MeOH (40
mL), then acetyl chloride (1.0 mL) was added to the
reaction mixture at 0 ꢀC. The solution was stoppered in
a flask and stirred at room temperature until TLC (3:1
petroleum ether–EtOAc) showed that the starting
material disappeared. The solution was neutralized with
Et3N, then concentrated to dryness. The residue was
passed through a short silica gel column to give 29 (715
mg, 66.8%) as a foamy solid. [a]D À17.5ꢀ (c 0.6,
Methyl (ꢀ-D-glucopyranosyluronic acid)-(1!2)-ꢁ-D-
mannopyranosyl-(1!3)-[ꢀ-D-xylopyranosyl-(1!2)-]ꢁ-D-
mannopyranosyl-(1!3)-[ꢀ-D-xylopyranosyl-(1!2)-]ꢁ-D-
mannopyranoside, ammonium salt (31). Hexasaccharide
30 (490 mg, 0.2 mmol) was dissolved in a satd metha-
nolic ammonia (50 mL). After 36 h at room tempera-
ture, water (1.0 mL) was added to the mixture to cleave
the methyl ester. After stirring at room temperature for
5 h, the reaction mixture was concentrated and purified
on a Bio-Gel P2 column (eluent: water), affording the
target hexasaccharide 31 (129 mg, 66.2%) as a foamy
solid. [a]D +99.6ꢀ (c 0.5, H2O); 1H NMR (D2O,
400 MHz): d 5.11 (s, 1H, H-1, Manp), 4.75 (s, 1H, H-1,
Manp), 4.31 (s, 1H, H-1, Manp), 4.29 (d, 1H, J1,2=8.0
Hz, H-1, GluAp), 4.12 (d, 1H, J1,2=8.9 Hz, H-1, Xylp),
4.10 (d, 1H, J1,2=9.2 Hz, H-1, Xylp), 3.33 (s, 3H,
OCH3); 13C NMR (100 MHz, D2O): 174.1 (–COONH4),
103.4, 103.3, 103.2, 102.5, 100.3, 100.2 (6 C-1), 79.0,
78.5, 78.4, 78.3, 78.3, 76.4, 76.2, 75.9, 75.8, 73.6, 73.4,
73.2, 72.9, 72.9, 70.7, 70.3, 69.5, 69.5, 68.4, 67.0, 66.7,
66.5, 65.4, 65.3, 61.5, 60.6, 60.6, 56.6 (O–CH3).
MALDI-TOF MS calcd for the ammonium salt of 31,
C35H61O30N: 975.8 [M]. Found: 975.8 (M);
980.9(MÀNH+4 +Na+).
1
CHCl3); H NMR (CDCl3, 400 MHz): d 8.12–7.34 (m,
65H, 13 PhH), 6.02 (dd, 1H, J3,4=J4,5=10.3 Hz, H-4,
Manp), 5.75 (dd, J1,2=J2,3=6.4 Hz, H-2, Xylp), 5.66 (dd,
1H, J2,3=3.2 Hz, J3,4=10.2 Hz, H-3, Manp), 5.57 (dd,
1H, J3,4=J4,5=10.0 Hz, H-4, Manp), 5.56 (dd, 1H,
J3,4=J4,5=9.7 Hz, H-4, Manp), 5.54–5.42 (m, 2H), 5.39
(m, 1H, H-4, Xylp), 5.32–5.29 (m, 2H), 5.22 (d, 1H,
J1,2=1.2 Hz, H-1, Manp), 4.98 (s, 1H, H-1, Manp), 4.82
(d, 1H, J1,2=4.8 Hz, H-1, Xylp), 4.53 (d, 1H, J1,2=1.7
Hz, H-1, Manp), 4.52 (d, 1H, J1,2=5.1 Hz, H-1, Xylp),
3.18 (s, 3H, OCH3). 13C NMR (100 MHz, CDCI3): 166.0,
165.9, 165.9, 165.6, 165.4, 165.4, 165.3, 165.2, 165.1,
165.0, 164.9, 164.6, 164.6 (13C, 13 COPh), 102.4, 99.9,
99.7, 99.3, 98.7(5C, 5 C-1), 54.8 (OCH3). Anal. calcd for
C120H102O37: C 67.47; H 4.81. Found: C 67.59; H 4.63.
Acknowledgements
Methyl [methyl 2,3,4-tri-O-acetyl-ꢀ-D-glucopyranosylur-
onate-(1!2)]-3,4,6-tri-O-benzoyl-ꢁ-D-mannopyranosyl-
(1!3)-[2,3,4-tri-O-benzoyl-ꢀ-D-xylopyranosyl-(1!2)-
]4,6-di-O-benzoyl-ꢁ-D-mannopyranosyl-(1!3)-[2,3,4-tri-
This work was supported by The Chinese Academy of
Sciences (KZCX3-J-08) and by The National Natural
Science Foundation of China (Projects 30070185 and
39970864).

J. Zhang, F. Kong / Bioorg. Med. Chem. 11 (2003) 4027–4037
4037
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