First syntheses of D-mannose penta- and decasaccharides, the repeating unit and its dimer of the cell-wall mannan of Candida kefyr IFO 0586

Article abstract of DOI:10.1016/S0957-4166(03)00216-7

α-D-Mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)- [α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)]- α-D-mannopyranose and α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→6)- [α-D-mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)]- α-D-mannopyranosyl-(1→6)-

Full text of DOI:10.1016/S0957-4166(03)00216-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 1275–1283
First syntheses of
D-mannose penta- and decasaccharides, the
repeating unit and its dimer of the cell-wall mannan of Candida
kefyr IFO 0586
Ying Xing and Jun Ning*
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871, Beijing 100085, PR China
Received 11 January 2003; revised 18 February 2003; accepted 26 February 2003
Abstract—a-
-mannopyranose and a-
osyl-(1“2)]-a- -mannopyranosyl-(1“6)-[a-
“6)-[a- -mannopyranosyl-(1“2)-a-
D
-Mannopyranosyl-(1“2)-a-
D
-mannopyranosyl-(1“6)-[a-
D
-mannopyranosyl-(1“2)-a-
-mannopyranosyl-(1“6)-[a- -mannopyranosyl-(1“2)-a-
-mannopyranosyl-(1
-mannopyranose, the repeating unit and its dimer of the cell
D
-mannopyranosyl-(1“2)]-a-
D
D
-mannopyranosyl-(1“2)-a-
D
D
D
-mannopyran-
D
D
-mannopyranosyl-(1“2)]-a-
D
D
D
-mannopyranosyl-(1“2)]-a-
D
wall mannan of the pathogenic yeast Candida kefyr IFO 0586, have been efficiently synthesized via their allyl glycosides by using
allyl 3,4,6-tri-O-benzoyl-a-
zoyl-a- -mannopyranoside as synthons. The blocked pentasaccharide was regio- and stereoselectively prepared by coupling of
allyl 3,4-di-O-benzoyl-a- -mannopyranoside with 2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl-(1“2)-6-O-acetyl-3,4-di-O-ben-
zoyl-a- -mannopyranosyl trichloroacetimidate, and then with 2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl-(1“2)-3,4,6-tri-O-
benzoyl-a- -mannopyranosyl trichloroacetimidate in a one-pot manner. © 2003 Elsevier Science Ltd. All rights reserved.
D
-mannopyranoside, allyl 6-O-acetyl-3,4-di-O-benzoyl-a-
D-mannopyranoside, and allyl 3,4-di-O-ben-
D
D
D
D
D
D
1. Introduction
natural oligosaccharides with a well-defined structure
and composition for biological studies aiming at a
better understanding of these phenomena at the molec-
ular level. Given the intrinsic difficulty in obtaining
complex natural oligosaccharides and glycoconjugates
in a pure and homogeneous form from natural sources
because of the presence of mixtures of glycosylated
species (glycoforms). A major opportunity is provided
by chemical synthesis.
Candida species are opportunistic pathogens of humans
that frequently cause severe systemic infections in
patients with AIDS,1 cancer,2 and burns3 as well as in
those under immunosuppressive or radiation therapy.4
An impressive feature of these fungi is that they synthe-
size cell wall polysaccharides containing predominantly
mannose residues.5–7 It has been reported that the
mannose oligosaccharides present in the fungal
nans deeply influence many fundamental biological
processes in the organisms. The a-linked oligo- -man-
D-man-
As part of our continuing efforts dedicated to develop-
ing efficient strategies for the construction of sugars, we
have prepared a lot of oligosaccharides with various
structures present in natural sources including fungi.12–16
In 1994, Kobayashi et al.17 conducted a structural
analysis of an antigenic cell wall mannan isolated from
the pathogenic yeast Candida kefyr IFO 0586 and
D
nosyl side chains of cell-wall mannan of the pathogenic
yeast Candida species play important roles in the bind-
ing of yeast cells to the marginal zone of mouse spleen8
and in the processes of several types of yeast floccula-
tion.9 The alkali-released a-linked
D-manno-oligosac-
charides obtained from a Candida albicans cell-wall
found that this sugar has a long a-(1“6)-linked
mannopyranosyl backbone and many short a-(1“2)-
linked -mannopyranosyl side-chains in a comb-like
D-
mannan were potent inhibitors of lymphoproliferation
induced by the parent
D
-mannan.10,11 Furthermore,
D
many of them (if not all) are antigenically relevant.
Hence, there is a great need for usable quantities of the
structure as shown in Figure 1. The reasons mentioned
above, together with the fact that there have been no
reports dealing with the synthesis of these highly
branched oligosaccharides, prompted us to develop an
efficient method for the synthesis of this kind of com-
* Corresponding author. Tel.: 8610-62849157; fax: 8610-62923563;
e-mail: jning@mail.rcees.ac.cn
plex carbohydrate. The preparation of allyl-a-D-penta-
0957-4166/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00216-7

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Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
Figure 1. The structures of the cell-wall mannan of Candida kefyr IFO 0586 and the synthesized oligosaccharides 1 and 2.
mannoside 1 and allyl-a-
D
-decamannoside 2 (Fig. 1),
Condensation of 3 and 5 with TMSOTf as catalyst and
,
the repeating unit and its dimer of the cell wall mannan
of the pathogenic yeast Candida kefyr IFO 0586 are
presented as typical examples using the strategy devel-
oped here.
4 A molecular sieves in CH2Cl2 at rt afforded the
disaccharides 10 in 88% yield. Deallylation of 10 with
PdCl2 followed by activation with CCl3CN in the pres-
ence of DBU, afforded the disaccharide donor 11 in
80% yield (for two steps) (Scheme 2). Similar proce-
dures gave another disaccharide donor 13.
2. Results and discussion
,
Coupling of 9 and 13 with TMSOTf as catalyst and 4 A
For the synthesis of the complex decasaccharide 2, we
needed 2,3,4,6-tetra-O-benzoyl-
molecular sieves in CH2Cl2 at −45°C regio- and
stereoselectively afforded the trisaccharides 14 in 91%
yield (Scheme 3). No (1“2)-linked trisaccharide was
D
-mannopyranosyl tri-
-manno-
chloroacetimidate 3, allyl 3,4,6-tri-O-benzoyl-
D
1
pyranoside 5, allyl 6-O-acetyl-3,4-di-O-benzoyl-a-
D
-
-
detected both from H NMR and TLC. Acetylation of
1
mannopyranoside 7, and allyl 3,4-di-O-benzoyl-
D
14 confirmed 6-O-glycosylation as the H NMR spec-
mannopyranoside 9 as building materials. Compound 3
was prepared according to the reported procedure.18
The glycosyl acceptor 5 was obtained from coupling of
trum of acetylated trisaccharide 15 showed a newly
emerged doublet of doublets at l 5.54 ppm for H-2.
Condensation of 14 with 11 afforded the blocked pen-
1
2-O-acetyl-3,4,6-tri-O-benzoyl-
D
-mannopyranosyl tri-
tasaccharide 16 in 90% yield. The H NMR data of 16
chloroacetimidate 418 with allyl alcohol followed by
2-O-deacetylation in 87% yield (Scheme 1). The glyco-
syl acceptors 7 and 9 were derived from 2,6-di-O-acetyl-
contained structurally characteristic information, i.e.
one acetyl signal (l 1.95), one allyl signal (l 5.43–5.27)
and five H-1 signals (l 5.60, 5.58, 5.39, 5.27, and 5.12).
The 13C NMR spectrum of 16 gave five signals for C-1
3,4-di-O-benzoyl-D-mannopyranosyl trichloroacetimi-
2
date 6 which was made according to the method devel-
oped in our laboratory.16 We were lucky to find that
coupling of 6 with allyl alcohol in the presence of
TMSOTf (0.3 equiv.) gave two compounds; one was the
required glycosyl acceptor 7 in 31%, and another was
(100.04, 99.65, 99.42, 98.36, 98.04) with JC1–H1 from
170 to 176.8 Hz indicating complete a-linkages.
Encouraged by the smooth coupling reaction results, a
one-pot procedure for the synthesis of the pentasaccha-
ride 16 was carried out. Thus, treatment of 9 and 13
,
allyl 2,6-di-O-acetyl-3,4-di-O-benzoyl-
D
-mannopyran-
with TMSOTf as catalyst and 4 A molecular sieves in
oside 8 in 63% yield. During the glycosylation process
of 6 with allyl alcohol, some of the 2-O-acetyl groups
of 6 were removed, and it was observed that with the
increase of the amount of TMSOTf added to the reac-
tion system, the 2-O-acetyl group removed product 7
was increased. Selective removal of the acetyl groups of
8 in MeOH containing 0.5% HCl gave the glycosyl
acceptor 9 in 93% yield.
CH2Cl2 at −45°C, followed by addition of 11 at rt
afforded compound 16 in 83% yield. Selective removal
of the 6-O-acetyl group of the pentasaccharide 16 gave
the glycosyl accepter 18. Deallylation of 16 with PdCl2
followed by activation with CCl3CN in the presence of
DBU gave the pentasaccharide donor 17. The fully
protected decasaccharide 19 was obtained by coupling
17 with 18 in 50% yield. The 1H NMR data of 19
Scheme 1. Reagents and conditions: (a) i. allyl alcohol (2 equiv.), TMSOTf (0.1 equiv.), CH2Cl2, rt, 1 h; ii. methanol/0.5% HCl,
rt, 12 h, 87% (over the two steps); (b) allyl alcohol (2 equiv.), TMSOTf (0.3 equiv), CH2Cl2, rt, 1 h, 31% for 7 and 63% for 8;
(c) methanol/0.5% HCl, rt, 12 h, 93%.

Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
1277
Scheme 2. Reagents and conditions: (a) CH2Cl2, TMSOTf (cat.), rt, 1 h, 88% for 10 and 92% for 12; (b) i. PdCl2, CH3OH, rt, 4
h; ii. CCl3CN, DBU, CH2Cl2, rt, 2 h, 80% for 11 and 81% for 13 (over the two steps).
Scheme 3. Reagents and conditions: (a) CH2Cl2, TMSOTf (cat.), rt, 1 h, 91% for 14, 90% for 16, 50% for 19; (b) Ac2O, pyridine,
,
rt, 3 h, 100%; (c) i. 13 (1.0 equiv.), TMSOTf (cat.), CH2Cl2, MS 4 A Powder, −45°C–rt, 1.5 h; ii. 11 (1.2 equiv.), rt, 1 h, 83% (over
the two steps); (d) i. PdCl2, CH3OH–CH2Cl2, 4 h; ii: CCl3CN, DBU, CH2Cl2 2 h, 85% (over the two steps); (e) methanol/0.5%
HCl, rt, 12 h, 92%; (f) CH3OH–CH2Cl2 (9:1) satd with dry NH3, 40°C, 24 h, 91% for 1, 95% for 2.
5.28, 5.24, 5.20, and 4.98). The 13C NMR spectrum of
contained structurally characteristic information, i.e.
19 gave ten signals for C-1 (100.07, 99.99, 99.72, 99.55,
99.43, 99.34, 98.89, 98.26, 98.19, 98.17). Deprotection
one acetyl signal (l 1.87), one allyl signal (l 5.36–5.20)
and ten H-1 signals (l 5.70, 5.62, 5.62, 5.48, 5.39, 5.32,

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Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
of 16 and 19 using NH3 in CH3OH–CH2Cl2 gave
compounds 1 and 2. The NMR spectra of the 1 and 2
are shown in Figures 2–5.
of the cell-wall mannan of the pathogenic yeast Can-
dida kefyr IFO 0586 was achieved by regio- and
stereoselective glycosylation using glycosyl trichloro-
imidates as the donors and partially protected
sugars as the acceptors. The sole use of acyl groups
in the synthesis substantially simplified the proce-
dure. This method should be useful for the synthesis
of other complex mannose oligosaccharides in the
future.
3. Conclusion
In summary, a highly efficient and concise synthesis
of the mannose pentasaccharide and decasaccharide
Figure 2. 1H NMR spectrum of compound 1 (solvent D2O at 25°C, 400 MHz Bruker ARX 400 spectrometer).
Figure 3. 13C NMR spectrum of compound 1 (solvent D2O at 25°C, 100 MHz Bruker ARX 400 spectrometer).
Figure 4. 1H NMR spectrum of compound 2 (solvent D2O at 25°C, 400 MHz Bruker ARX 400 spectrometer).

Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
1279
Figure 5. 13C NMR spectrum of compound 2 (solvent D2O at 25°C, 100 MHz Bruker ARX 400 spectrometer).
4. Experimental
4.1. General methods
layer was dried over Na2SO4 and concentrated to a
syrup. Purification of the residue by flash chromatogra-
phy (2:1 petroleum ether–EtOAc) gave 5 as a syrup (2.9
1
g, 87% for the two steps): [h]D −15.1 (c 1.0, CHCl3); H
Optical rotations were determined at 25°C with a
Perkin-Elmer Model 241-Mc automatic polarimeter.
Melting points were determined with a ‘Mel-Temp’
apparatus. 1H NMR and 13C NMR spectra were
recorded with Bruker ARX 400 spectrometers (400
NMR (CDCl3, 400MHz): l 8.03–7.33 (m, 15 H, 3
PhH), 5.95 (m, 1 H, CH=CH2), 5.94 (dd, 1 H, J3,4
=
J
4,5=9.8 Hz, H-4), 5.70 (dd, 1 H, J2,3=3.1 Hz, J4,5=9.8
2
3
Hz, H-3), 5.33 (dd, 1H, J=1.5 Hz, Jtrans=17.2 Hz,
2
3
CH=CH2), 5.24 (dd, 1 H, J=1.5 Hz, Jcis=10.4 Hz,
CH=CH2), 5.03 (d, 1 H, J1,2=1.6 Hz, H-1), 4.58 (dd,
1 H, H-6), 4.48 (dd, 1 H, H-6%), 4.40–4.08 (m, 4 H,
CH2CH=CH2, H-2, 5). Anal. calcd for C30H28O9: C,
67.66; H, 5.30. Found: C, 67.93; H, 5.27.
1
MHz for H, 100 MHz for 13C) for solutions in CDCl3
or D2O as indicated. Chemical shifts are given in ppm
downfield from internal Me4Si. Mass spectra were mea-
sured using MALTI-TOF-MS with CCA as matrix.
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
detector. Column chromatography was conducted by
elution of a column (16×240 mm, 18×300 mm, 35×400
mm) of silica gel (100–200 mesh) with EtOAc-
petroleum ether (60–90°C) as the eluent. Solutions were
concentrated at <60°C under reduced pressure.
4.3. Allyl 6-O-acetyl-3,4-di-O-benzoyl-a-D-mannopyran-
oside 7 and Allyl 2,6-di-O-acetyl-3,4-di-O-benzoyl-a-D-
mannopyranoside 8
A
solution of 2,6-di-O-acetyl-3,4-di-O-benzoyl-a-D-
mannopyranosyl trichloroacetimidate 6 (11 g, 17.8
mmol) and allyl alcohol (2.4 mL, 35 mmol) in CH2Cl2
(200 mL) was stirred with dried molecular sieves (4 A,
,
4.2. Allyl 3,4,6-tri-O-benzoyl-a-
D
-mannopyranoside 5
4 g) under N2 for 15 min, and then TMSOTf (1.0 mL,
5.3 mmol) was added. After 1 h, the reaction mixture
was neutralized with Et3N, filtered and the filtrate was
concentrated. Purification of the residue by chromatog-
raphy (3:1 petroleum ether–EtOAc) gave allyl 6-O-ace-
A
solution of 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-
mannopyranosyl trichloroacetimidate 4 (4.3 g, 6.4
mmol) and allyl alcohol (0.7 ml, 10 mmol) in dry
CH2Cl2 (50 mL) was stirred with dried molecular sieves
tyl-3,4-di-O-benzoyl-a- -mannopyranoside 7 (2.6 g,
D
,
(4 A, 1 g) under N2 for 15 min, and then TMSOTf (135
31%) and allyl 2,6-di-O-acetyl-3,4-di-O-benzoyl-a-D-
mL, 0.7 mmol) was added. After 1 h, the reaction
mixture was neutralized with Et3N, filtered and the
filtrate was concentrated. The resulting residue without
purification was directly dissolved in MeOH (150 mL)
containing 0.5% HCl. The solution was stirred at rt for
12 h, at the end of which time TLC (2:1 petroleum
ether–EtOAc) indicated that the starting material had
disappeared. The mixture was neutralized with Et3N
and then concentrated to dryness. The residue was
partitioned between water and CH2Cl2, the organic
mannopyranoside 8 (5.7 g, 63%). For 7: [h]D −13.4 (c
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l 7.97–7.26
(m, 10 H, 2 BzH), 5.98 (m, 1 H, CH=CH2), 5.85 (dd,
1H, J3,4=J4,5=10 Hz, H-4), 5.65 (dd, 1 H, J2,3=3.0 Hz,
2
3
J
3,4=10 Hz, H-3), 5.37 (dd, 1H, J=1.5 Hz, Jtrans=
2 3
17.2 Hz, CH=CH2), 5.27 (dd, 1 H, J=1.5 Hz, Jcis=
10.4 Hz, CH=CH2), 5.00 (d, 1 H, J1,2=1.5 Hz, H-1),
4.34–4.19 (m, 5 H, CH2CH=CH2, H-2, H-5, H-6), 4.10
(dd, 1 H, J5,6%=6.1 Hz, J6,6%=12.8 Hz, H-6%), 2.05 (s, 3
H, COCH3). Anal. calcd for C25H26O9: C, 63.82; H,

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Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
5.57. Found: C, 63.61; H, 5.53. For 8: [h]D −17.5 (c 0.5,
mmol) and DBU (100 mL, 0.71 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 reac-
tion mixture followed by purification on a silica gel
column with 2:1 petroleum ether-EtOAc as the eluent
furnished the disaccharide donor 11 (3.34 g, 80% two
1
CHCl3); H NMR (400 MHz, CDCl3): l 7.96–7.33 (m,
10 H, 2 BzH), 5.96 (m, 1 H, CH=CH2), 5.78–5.76 (m,
2
2 H, H-3, 4), 5.48 (dd, 1 H, H-2), 5.36 (dd, 1H, J=1.3
3
2
Hz, Jtrans=17.1 Hz, CH=CH2), 5.28 (dd, 1 H, J=1.3
3
Hz, Jcis=10.4 Hz, CH=CH2), 4.99 (d, 1 H, J1,2=1.7
Hz, H-1), 4.37-4.09 (m, 5 H, CH2CH=CH2, H-5, 6, 6%),
2.16, 2.07 (2s, 6 H, 2 COCH3). Anal. calcd for
C27H28O10: C, 63.28; H, 5.51. Found: C, 63.03; H, 5.47.
1
steps): [h]D −24.5 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): l 8.72 (s, 1 H, NH), 8.09–7.26 (m, 35 H, 7
BzH), 6.64 (d, 1 H, J2,1=1.9 Hz, H-1), 6.19-5.93 (m, 5
H, 2 H-4, 2 H-3, 1 H-2), 5.37 (d, 1 H, J1%,2%=1.5 Hz,
H-1), 4.75–4.48 (m, 7 H, H-2, 2 H-5, 4 H-6). Anal.
calcd for C63H50Cl3NO18: C, 62.26; H, 4.15. Found: C,
62.08; H, 4.19.
4.4. Allyl 3,4-di-O-benzoyl-a-D-mannopyranoside 9
A solution of 8 (5.6 g, 10.9 mmol) in MeOH (160 mL)
containing 0.5% HCl was stirred at rt for 12 h, at the
end of which time TLC (2:1 petroleum ether-EtOAc)
indicated that the starting material had disappeared.
The mixture was neutralized with Et3N and then con-
centrated to dryness. The residue was partitioned
between water and CH2Cl2, the organic layer was dried
over Na2SO4 and concentrated to a syrup. Purification
of the residue by flash chromatography (2:1 petroleum
ether–EtOAc) gave 9 as a syrup (4.4 g, 93%): [h]D −18.6
4.7. Allyl 2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl-
(1“2)-6-O-acetyl-3,4-di-O-benzoyl-a-D-
mannopyranoside 12
A solution of 7 (1.5 g, 3.19 mmol) and 2,3,4,6-tetra-O-
benzoyl-a- -mannopyranosyl trichloroacetimidate (3)
(2.60 g, 3.51 mmol) in dry CH2Cl2 (40 mL) was stirred
D
1
(c 2.0, CHCl3); H NMR (CDCl3, 400MHz): l 8.00–
,
with dried molecular sieves (4 A, 1 g) under N2 for 15
7.32 (m, 10 H, 2 PhH), 5.97 (m, 1 H, CH=CH2),
5.82–5.75 (m, 2 H, H-3, 4), 5.39–5.27 (m, 2 H, CH=
CH2), 5.05 (d, 1 H, J1,2=1.6 Hz, H-1), 4.31-3.70 (m, 6
H, CH2CH=CH2, H-2, 5, 6, 6%). Anal. calcd for
C23H24O8: C, 64.48; H, 5.65. Found: C, 64.23; H, 5.68.
min, and then TMSOTf (50 mL) was added. After 1 h,
the reaction mixture was neutralized with Et3N, filtered
and the filtrate was concentrated. Purification of the
residue by flash chromatography (3:1 petroleum ether–
EtOAc) gave 12 as a syrup (3.35 g, 92%): [h]D −45.3 (c
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l 8.01–7.26
4.5. Allyl 2,3,4,6-tetra-O-benzoyl-a-
D-mannopyranosyl-
(m, 30 H, 6 BzH), 6.10–5.86 (m, 6 H, CH=CH2, 2 H-4,
(1“2)-3,4,6-tri-O-benzoyl-a- -mannopyranoside 10
D
2
3
2 H-3, H-2), 5.35-5.31 (dd, 1 H, J=1.5 Hz, Jtrans
=
2
A solution of allyl 3,4,6-tri-O-benzoyl-a-
ranoside 5 (2.90 g, 5.45 mmol) and 2,3,4,6-tetra-O-ben-
zoyl-a- -mannopyranosyl trichloroacetimidate 3 (4.44
D-mannopy-
17.2 Hz, CH=CH2), 5.27–5.24 (dd, 1 H, J=1.3 Hz,
3Jcis=10.4 Hz, CH=CH2), 5.25 (d, 1 H, J2,1=1.7 Hz,
H-1), 5.21 (d, 1 H, J2,1=1.7 Hz, H-1), 4.72-4.01 (m, 9
H, CH2CH=CH2, H-2, 2 H-5, 4 H-6), 2.17 (s, 3 H,
COCH3). Anal. calcd for C59H52O18: C, 67.55; H, 5.00.
Found: C, 67.75; H, 5.04.
D
g, 6.00 mmol) in dry CH2Cl2 (60 mL) was stirred with
,
dried molecular sieves (4 A, 2 g) under N2 for 15 min,
and then TMSOTf (105 mL, 0.55 mmol) was added.
After 1 h, the reaction mixture was neutralized with
Et3N, filtered and the filtrate was concentrated. Purifi-
cation of the residue by flash chromatography (3:1
petroleum ether-EtOAc) gave 10 as a syrup (5.33 g,
4.8. ,3,4,6-Tetra-O-benzoyl-a-
D
-mannopyranosyl-(1“2)-
6-O-acetyl-3,4-di-O-benzoyl-a-
D-mannopyranosyl
trichloroacetimidate 13
1
88%): [h]D −37.9 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): l 8.10–7.20 (m, 35 H, 5 BzH), 6.14–5.86 (m, 6
H, CH=CH2, 2 H-4, 2 H-3, H-2), 5.31 (dd, 1 H,
A mixture of compound 12 (2.30 g, 2.19 mmol) and
PdCl2 (70 mg) in MeOH (100 mL) was stirred vigor-
ously for 4 h at rt, TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The reaction
mixture was filtered through Celite, and the filtrate was
concentrated to dryness. The resulting compound was
dissolved in CH2Cl2 (30 mL), then CCl3CN (1.0 mL, 10
mmol) and DBU (50 mL) 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 on a silica gel column
with 2:1 petroleum ether–EtOAc as the eluent, fur-
nished the disaccharide donor 13 (2.05 g, 81% two
3
2J=1.4 Hz, Jtrans=18.5 Hz, CH=CH2), 5.22 (dd, 1 H,
3
2J=1.3 Hz, Jcis=10.4 Hz, CH=CH2), 5.28 (d, 1 H,
J
1,2=1.6 Hz, H-1), 5.22 (d, 1 H, J1,2=1.6 Hz, H-1),
4.69–4.40 (m, 7 H, H-2, 2 H-5, 4 H-6), 4.27-3.99 (m, 2
H, CH2CH=CH2). Anal. calcd for C64H54O18: C,
69.18; H, 4.90. Found: C, 68.98; H, 4.99.
4.6. 2,3,4,6-Tetra-O-benzoyl-a-
D
-mannopyranosyl-(1“
2)-3,4,6-tri-O-benzoyl-a-
D-mannopyranosyl trichloro-
acetimidate 11
A mixture of compound 10 (3.82 g, 3.44 mmol) and
PdCl2 (100 mg) in MeOH (150 mL) was stirred vigor-
ously for 4 h at rt, TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The reaction
mixture was filtered through Celite and the filtrate was
concentrated to dryness. The resulting compound was
dissolved in CH2Cl2 (40 mL), then CCl3CN (1.5 ml, 15
1
steps): [h]D −43.7 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): l 8.73 (s, 1 H, NH), 8.09–7.26 (m, 30 H, 6
BzH), 6.65 (d, 1 H, J2,1=1.7 Hz, H-1), 6.19-5.89 (m, 5
H, 2 H-4, 2 H-3, H-2), 5.35 (d, 1 H, J2%,1%=1.3 Hz, H-1),
4.80–4.34 (m, 7 H, H-2, 2 H-5, 4 H-6), 2.17 (s, 3 H,

Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
1281
COCH3). Anal. calcd for C58H48Cl3NO18: C, 60.40; H,
4.19. Found: C, 60.61; H, 4.16.
and then TMSOTf (20 mL) was added. After 1 h, the
reaction mixture was neutralized with Et3N, filtered and
the filtrate was concentrated. Purification of the residue
by column chromatography (1.5:1 petroleum ether–
EtOAc) gave 16 as a syrup (2.87 g, 90%): [h]D −45.2 (c
4.9. 2,3,4,6-Tetra-O-benzoyl-a-
2)-6-O-acetyl-3,4-di-O-benzoyl-a-
“6)-3,4-di-O-benzoyl-a- -mannopyranoside 14
D
-mannopyranosyl-(1“
D
-mannopyranosyl-(1
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l 8.00–6.93
D
(m, 75 H, 15 BzH), 6.20–5.75 (m, 13 H, CH=CH2, 5
H-4, 5 H-3, 2 H-2), 5.60 (d, 1 H, J1,2=1.6 Hz, H-1),
A solution of 13 (1.76 g, 1.53 mmol) and 3,4-di-O-ben-
zoyl-a- -mannopyranoside 9 (647 mg, 1.51 mmol) in
2
5.58 (d, 1 H, J1,2=1.6 Hz, H-1), 5.41 (dd, 1 H, J=1.5
D
3
Hz, Jtrans=18.7 Hz, CH=CH2), 5.39 (d, 1 H, J1,2=1.6
dry CH2Cl2 (30 mL) was stirred with dried molecular
2
3
Hz, H-1), 5.28 (dd, 1 H, J=1.3 Hz, Jcis=11.8 Hz,
CH=CH2), 5.27 (d, 1 H, J1,2=1.6 Hz, H-1), 5.12 (d, 1
H, J1,2=1.6 Hz, H-1), 1.95 (s, 3 H, COCH3). 13C NMR
(100 MHz, CDCl3): l 170.66 (1 C, COCH3), 166.17,
166.03, 165.70, 165.59, 165.59, 165.59, 165.44, 165.19,
165.19, 165.19, 165.13, 164.94, 164.94, 164.66, 164.65
(15 C, 15 COPh), 133.4-128.0 (91 C, 15 Ph, CH2CH=
CH2), 117.80 (CH2CH=CH2), 100.04, 99.65, 99.42,
98.36, 98.04 (5 C-1, 2JC1-H1=170-176.8 Hz), 78.95,
77.66, 77.20 (3 C-2), 20.5 (1 C, COCH3). Anal. calcd
for C140H118O42: C, 68.01; H, 4.81. Found: C, 68.29; H,
4.77.
,
sieves (4 A, 1 g) under N2 for 15 min, and then
TMSOTf (30 mL) was added. After 1 h, the reaction
mixture was neutralized with Et3N, filtered and the
filtrate was concentrated. Purification of the residue by
column chromatography (2:1 petroleum ether-EtOAc)
gave 14 as a syrup (1.96 g, 91%): [h]D −33.2 (c 1.0,
1
CHCl3); H NMR (400 MHz, CDCl3): l 8.08–7.32 (m,
40 H, 8 BzH), 6.13–5.72 (m, 8 H, CH=CH2, H-2, 3
H-3, 3 H-4), 5.44 (dd, 1 H, 2J=1.5 Hz, 3Jtrans=17.2 Hz,
2
3
CH=CH2), 5.28 (dd, 1 H, J=1.3 Hz, Jcis=10.5 Hz,
CH=CH2), 5.29 (d, 1 H, J1,2=1.5 Hz, H-1), 5.23 (d, 1
H, J1,2=1.5 Hz, H-1), 5.05 (d, 1 H, J1,2=1.5 Hz, H-1),
4.76 (dd, 1 H, H-6), 4.65 (dd, 1 H, H-6), 4.46–4.10 (m,
9 H, CH2CH=CH2, 3 H-5, 2 H-6, 2 H-2), 3.90 (dd, 1
H, H-6), 3.71 (dd, 1 H, H-6), 2.11 (s, 3 H, COCH3).
Anal. calcd for C79H70O25: C, 66.85; H, 4.97; Found: C,
67.07; H, 4.92.
4.12. A one-pot procedure for the preparation of the
pentasaccharide 16
A solution of 9 (0.9 g, 2.10 mmol) and 13 (2.45 g, 2.13
mmol) in dry CH2Cl2 (100 mL) was stirred with acti-
,
vated 4 A molecular sieves (2 g) at rt under N2 for 20
4.10. 2,3,4,6-Tetra-O-benzoyl-a-
D
-mannopyranosyl-(1“
-mannopyranosyl-(1
-mannopyranoside
min. Then the reaction mixture was cooled to −45°C,
and TMSOTf (10 mL) was added. After 30 min, temper-
ature was allowed to rise to rt. After the reaction
mixture was stirred for further 1 h, to the reaction
mixture was added 11 (2.83 g, 2.33 mmol) under N2 at
rt. The reaction mixture was stirred for 1 h, at the end
of which time TLC (2:1 petroleum ether–EtOAc) indi-
cated that the reaction was complete. The reaction
mixture was neutralized with Et3N, filtered and the
filtrate was concentrated. Purification of the resultant
residue by column chromatography with 2:1 petroleum
ether–EtOAc as eluent gave 16 (4.7 g, 83%).
2)-6-O-acetyl-3,4-di-O-benzoyl-a-
D
“6)-2-O-acetyl-3,4-di-O-benzoyl-a-
15
D
To a solution of 14 (1.20 g, 0.84 mmol) in pyridine (20
mL) Ac2O (2 mL) was added. After the mixture was
stirred for 3 h at rt, it was diluted with CH2Cl2, washed
with 1 N HCl, water, and satd aq NaHCO3. The
organic layers were combined, dried, and concentrated.
Purification by column chromatography (2:1 petroleum
ether–EtOAc) quantitatively gave 15 as a syrup: [h]D
1
−29.3 (c 1.5, CHCl3); H NMR (400 MHz, CDCl3): l
8.03–7.27 (m, 40 H, 8 BzH), 6.14–5.79 (m, 8 H, CH=
4.13. 2,3,4,6-Tetra-O-benzoyl-a-
2)-6-O-acetyl-3,4-di-O-benzoyl-a-
“6)-[2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl-(1“
-mannopyranosyl-(1“2)]-3,4-
-mannopyranosyl trichloroacetimidate
D
-mannopyranosyl-(1“
CH2, H-2, 3 H-3, 3 H-4), 5.53 (dd, 1 H, H-2), 5.46 (dd,
2
3
D-mannopyranosyl-(1
1 H, J=1.5 Hz, Jtrans=17.2 Hz, CH=CH2), 5.31 (dd,
2
3
D
1 H, J=1.3 Hz, Jcis=10.5 Hz, CH=CH2), 5.23 (d, 1
H, J1,2=1.5 Hz, H-1), 5.20 (d, 1 H, J1,2=1.5 Hz, H-1),
5.02 (d, 1 H, J1,2=1.5 Hz, H-1), 4.73 (dd, 1 H, H-6),
4.62 (dd, 1 H, H-6), 4.47–4.09 (m, 8 H, CH2CH=CH2,
3 H-5, 2 H-6, H-2), 3.92 (dd, 1 H, H-6), 3.65 (dd, 1 H,
H-6), 2.19, 2.08 (2s, 6 H, 2 COCH3). Anal. calcd for
C81H72O26: C, 66.57; H, 4.97; Found: C, 66.34; H, 5.01.
2)-3,4,6-tri-O-benzoyl-a-
D
di-O-benzoyl-a-
D
17
A mixture of compound 16 (1.00 g, 0.36 mmol) and
PdCl2 (30 mg) in MeOH (50 mL) was stirred vigorously
for 4 h at rt, TLC (1.5:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The reaction
mixture was filtered through Celite and the filtrate was
concentrated to dryness. The resulting compound was
dissolved in CH2Cl2 (15 mL), then CCl3CN (0.5 ml, 5
mmol) and DBU (50 mL) 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 on a silica gel column
with 2:1 petroleum ether–EtOAc as the eluent furnished
4.11. Allyl 2,3,4,6-tetra-O-benzoyl-a-
(1“2)-6-O-acetyl-3,4-di-O-benzoyl-a-
syl-(1“6)-[2,3,4,6-tetra-O-benzoyl-a-
(1“2)-3,4,6-tri-O-benzoyl-a-
3,4-di-O-benzoyl-a- -mannopyranoside 16
D-mannopyranosyl-
D
-mannopyrano-
D
-mannopyranosyl-
D
-mannopyranosyl-(1“2)]-
D
A solution of 14 (1.65 g, 1.16 mmol) and 11 (1.56 g,
1.28 mmol) in dry CH2Cl2 (30 mL) was stirred with
,
dried molecular sieves (4 A, 1.5 g) under N2 for 15 min,

1282
Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
the pentasaccharide donor 17 (798 mg, 85% two
steps): [h]D −35.0 (c 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): l 8.76 (s, 1 H, NH), 8.05–6.81 (m, 75
H, 15 BzH), 6.55 (d, 1 H, J1,2 1.6 Hz, H-1), 6.16–
5.87 (m, 12 H, 5 H-4, 5 H-3, 2 H-2), 5.70 (d, 1 H,
(d, 1 H, J1,2=1.3 Hz, H-1), 5.39(d, 1 H, J1,2=1.3 Hz,
H-1), 5.34 (dd, 1 H, 2J=1.3 Hz, 3Jtrans=17.1 Hz,
CH=CH2), 5.32 (d, 1 H, J1,2=1.3 Hz, H-1), 5.28 (d,
1 H, J1,2=1.3 Hz, H-1), 5.24 (d, 1 H, J1,2=1.3 Hz,
2
3
H-1), 5.22 (dd, 1 H, J=1.3 Hz, Jcis=10.4 Hz, CH=
CH2), 5.20 (d, 1 H, J1,2=1.3 Hz, H-1), 4.98 (d, 1 H,
J1,2=1.3 Hz, H-1), 4.93–3.26 (m, 38 H, CH2CH=
CH2, 10 H-5, 20 H-6·6 H-2), 1.87 (s, 3 H, COCH3);
J
1,2=1.3 Hz, H-1), 5.63 (d, 1 H, J1,2=1.3 Hz, H-1),
5.38 (d, 1 H, J1,2=1.3 Hz, H-1), 5.36 (d, 1 H, J1,2
1.3 Hz, H-1), 4.99–3.60 (m, 18 H, 3 H-2, 5 H-5, 10
H-6), 1.93 (s, H, COCH3). Anal. calcd for
139H114Cl3NO42: C, 64.79; H, 4.46. Found: C, 64.56;
H, 4.50.
=
3
13C NMR (100 MHz, CDCl3):
l 170.69 (1 C,
C
COCH3), 166.2-164.5 (30 C, 30 COPh), 133.4–127.5
(181 C, 30 Ph, CH=CH2), 117.7 (CH=CH2), 100.07,
99.99, 99.72, 99.55, 99.43, 99.34, 98.89, 98.26, 98.19,
98.17 (10 C-1), 20.4 (1 C, COCH3). Anal. calcd for
C275H228O82: C, 68.18; H, 4.74. Found: C, 68.32; H,
4.80.
4.14. Allyl 2,3,4,6-tetra-O-benzoyl-a-
(1“2)-3,4-di-O-benzoyl-a- -mannopyranosyl-(1“6)-
[2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl-(1“2)-
3,4,6-tri-O-benzoyl-a- -mannopyranosyl-(1“2)]-
3,4-di-O-benzoyl-a- -mannopyranoside 18
D-mannopyranosyl-
D
D
D
4.16. Allyl a-
pyranosyl-(1“6)-[a-
mannopyranosyl-(1“2)]-a-
D
-mannopyranosyl-(1“2)-a-
-mannopyranosyl-(1“2)-a-
-mannopyranoside 1
D-manno-
D
D
D-
D
A solution of 16 (1.2 g, 0.44 mmol) in MeOH (100
mL) containing 0.5% HCl was stirred at rt for 12 h,
at the end of which time TLC (1.5:1 petroleum ether–
EtOAc) indicated that the starting material had disap-
peared. The mixture was neutralized with Et3N, and
then concentrated to dryness. The residue was parti-
tioned between water and CH2Cl2, the organic layer
was dried over Na2SO4, and concentrated to a syrup.
Purification of the residue by column chromatography
(1.5:1 petroleum ether–EtOAc) gave 18 as a syrup
Compound 16 (758 mg, 0.28 mmol) was dissolved in
an ammonia-saturated solution of 1:9 CH2Cl2–
CH3OH (100 mL) at 40°C. After 24 h, the reaction
mixture was concentrated to about 10 mL, and then
CH2Cl2 (100 mL) was added. The resultant precipi-
tate was filtered and washed four times with CH2Cl2
to afford 1 as white solid (219 mg, 91%): [h]D +12.3
1
(c 1.0, H2O); H NMR (D2O, 400 MHz): l 5.85 (m,
1
1 H, CH=CH2), 5.27–5.17 (m, 2 H, CH=CH2), 5.19,
5.03, 5.02, 4.94, 4.93 (5s, 5 H, 5 H-1); 13C NMR (100
MHz, D2O): 133.52 (1 C, CH2CH=CH2), 118.71 (1
C, CH2CH=CH2), 102.23, 102.23, 100.7, 98.12, 97.50
(5 C-1). MALDI-TOF MS calcd for C33H56O26:
868.79 [M]. found: 891.65 (M+Na)+.
(978 mg, 92%): [h]D −62.7 (c 1.0, CHCl3); H NMR
(400 MHz, CDCl3): l 8.03–6.96 (m, 75 H, 15 BzH),
6.13–5.71 (m, 13 H, CH=CH2, 5 H-4, 5 H-3, 2 H-2),
5.56 (d, 1 H, J1,2=1.2 Hz, H-1), 5.51 (d, 1 H, J1,2
=
2
3
1.2 Hz, H-1), 5.40 (dd, 1 H, J=1.5 Hz, Jtrans=17.2
Hz, CH=CH2), 5.31 (d, 1 H, J1,2=1.2 Hz, H-1), 5.29
3
(dd, 1 H, 2J=1.3 Hz, Jcis=10.4 Hz), 5.25 (d, 1 H,
4.17. Allyl ˜-
pyranosyl-(1“6)-[a-
mannopyranosyl-(1“2)]-a-
[a- -mannopyranosyl-(1“2)]-a-
(1“6)-[a- -mannopyranosyl-(1“2)-a-
pyranosyl-(1“2)]-a- -mannopyranoside 2
D
-mannopyranosyl-(1“2)-a-
-mannopyranosyl-(1“2)-a-
-mannopyranosyl-(1“6)-
-mannopyranosyl-
-manno-
D-manno-
J
1,2=1.2 Hz, H-1), 5.08 (d, 1 H, J1,2=1.2 Hz, H-1),
D
D-
4.86–3.41 (m, 20 H,CH2CH=CH2, 3 H-2, 5 H-5, 10
H-6). Anal. calcd for C138H116O41: C, 68.20; H, 4.81.
Found: C, 68.41; H, 4.71.
D
D
D
D
D
D
4.15. Allyl 2,3,4,6-tetra-O-benzoyl-a-
(1“2)-6-O-acetyl-3,4-di-O-benzoyl-a-
syl-(1“6)-[2,3,4,6-tetra-O-benzoyl-a-
(1“2)-3,4,6-tri-O-benzoyl-a-
3,4-di-O-benzoyl-a-
tetra-O-benzoyl-a-
D-mannopyranosyl-
D
-mannopyrano-
D
-mannopyranosyl-
Compound 19 (386 mg, 0.08 mmol) was dissolved in
an ammonia-saturated solution of 1:9 CH2Cl2–
CH3OH (120 mL) at 40°C. After 24 h, the reaction
mixture was concentrated to about 30 mL, and then
CH2Cl2 (100 mL) was added. The resultant precipi-
tate was filtered and washed four times with CH2Cl2
to afford 2 as white solid (127 mg, 95%): [h]D +35.8
D
-mannopyranosyl-(1“2)]-
-mannopyranosyl-(1“6)-[2,3,4,6-
D
D
-mannopyranosyl-(1“2)]-3,4-di-O-
benzoyl-a-
benzoyl-a-
benzoyl-a-
D
D
D
-mannopyranosyl-(1“6)-[2,3,4,6-tetra-O-
-mannopyranosyl-(1“2)-3,4,6-tri-O-
-mannopyranosyl-(1“2)]-3,4-di-O-benzoyl-
1
a-D-mannopyranoside 19
(c 1.0, H2O); H NMR (D2O, 400 MHz): l 5.94 (m,
1 H, CH=CH2), 5.35–5.25 (2 H, CH=CH2), 5.25–
5.02 (10 H-1); 13C NMR (100 MHz, D2O): 133.6 (1
C, CH2CH=CH2), 118.7 (1 C, CH2CH=CH2),
101.97, 101.97, 101.97, 101.97, 100.46, 100.38, 98.11,
97.96, 97.96, 97.39 (10 C-1). MALDI-TOF MS calcd
for C63H106O51: 1679.50 [M]. found: 1702.61 (M+Na)+
.
A solution of 17 (550 mg, 0.21 mmol) and 18 (461
mg, 0.19 mmol) in dry CH2Cl2 (20 mL) was stirred
,
with dried molecular sieves (4 A, 0.5 g) under N2 for
15 min, and then TMSOTf (5 mL) was added. After 1
h, the reaction mixture was neutralized with Et3N,
filtered and the filtrate was concentrated. Purification
of the residue by a column chromatography (1.3:1
petroleum ether–EtOAc) gave 19 as a syrup (460 mg,
1
50%): [h]D −33.7 (c 1.0, CHCl3); H NMR (400 MHz,
Acknowledgements
CDCl3): l 8.02–6.60 (m, 150 H, 30 BzH), 6.40–5.82
(m, 25 H, CH=CH2 10 H-4, 10 H-3, 4 H-2), 5.70 (d,
1 H, J1,2=1.3 Hz, H-1), 5.62 (m, 2 H, 2 H-1), 5.48
This work was supported by the Beijing Natural Sci-
ence Foundation (6021004).

Y. Xing, J. Ning / Tetrahedron: Asymmetry 14 (2003) 1275–1283
1283
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