Facile synthesis of arabinomannose penta- and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Article abstract of DOI:10.1016/j.carres.2004.04.012

Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer 31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor 13 with a

Full text of DOI:10.1016/j.carres.2004.04.012

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1761–1771
Facile synthesis of arabinomannose penta- and decasaccharide
fragments of the lipoarabinomannan of
the equine pathogen, Rhodococcus equi
Zuchao Ma, Jianjun Zhang and Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Academia Sinica, PO Box 2871, Beijing 100085, PR China
Received 10 March 2004; accepted 15 April 2004
Available online 7 June 2004
Abstract—Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer
31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor
13 with a free hydroxyl group at C-20 of the mannose residue attached to the core mannose residue by (1 ! 6)-linkage, followed by
coupling with 2,3,5-tri-O-benzoyl-a-
D
-arabinofuranosyl-(1 ! 2)-3,4,6-tri-O-benzoyl-a-
D-mannopyranosyl trichloroacetimidate (18),
and by deacylation. Meanwhile, the decamer 31 was obtained by firstly preparing a benzoylated mannose (1 ! 6)-linked tetra-
saccharide backbone 26 with 2-, 200-O-ClAc, and 20-, 2000-O-Ac groups, respectively, then by dechloroacetylation and subsequent
condensation with perbenzoylated trichloroacetimidate, and then by deacetylation and subsequent coupling with 18, and finally, by
deacylation.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Mannose; Arabinose; Regio- and stereoselective synthesis
1. Introduction
losis4 LAM has also been reported to have powerful
immunomodulatory properties, promoting distinctive
patterns of macrophages cytokine induction that sub-
sequently directs host immune responses.5 Small differ-
ences in LAM structure can strongly influence these
biological activities. The lipoglycan of R. equi has been
isolated, purified, and characterized,6 and its structure is
shown in Figure 1. It is composed of three domains. The
first one is a polysaccharide consisting of a comb-like,
2-Manp or -Araf-(1 ! 2)-Manp branched six-linked
mannose pentasaccharide repeating unit. The second
one is a polysaccharide consisting of a mannose (1 ! 6)-
linked disaccharide repeating unit, and the third one is
the phosphatidyl-myo-inositol anchor attached with di-
acylated Manp. The synthesis of (1 ! 6)-linked mannose
hexa-, octa-, and dodecasaccharide, corresponding to
the structure of the second domain, has been reported
by our group.7 We present herein the synthesis of the
pentasaccharide repeating unit and its dimer, corre-
sponding to the structure of the first domain of R. equi
LAM.
The equine pathogen, Rhodococcus equi, is a significant
cause of disease in foals between the age of 1 and
5 months and is responsible for around 3% of global foal
mortality.1 This organism has also emerged as an
opportunistic human pathogen, notably of people with
compromised immunity.2 R. equi is an intracellular
pathogen of alveolar macrophages, a member of the
mycolata, and its infection is characterized by bron-
chopneumonia. Members of the mycolata have a char-
acteristic cell envelope that profoundly affects the
properties of these bacteria, and its composition and
organization have been a major focus of mycobacterial
research.3 Lipoarabinomannan (LAM) is a complex
mycobacterial cell envelope component that has been
identified as a putative virulence factor of M. tubercu-
* Corresponding author. Tel.: +86-10-62936613; fax: +86-10-629235-
63; e-mail: fzkong@mail.rcees.ac.cn
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.04.012

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Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
Figure 1. Structure of R. equi LAM.
2. Results and discussion
13C NMR spectra of 20 showed all of the characteristic
signals such as at d 5.06 (s, 1H, Manp H-1), 5.05 (s, 1H,
Araf H-1), 5.01 (s, 1H, Manp H-1), 4.99 (s, 1H, Manp
H-1), 4.89 (s, 1H, Manp H-1); 109.36 (Araf C-1), 102.32,
101.36, 97.95, and 97.47 (Manp C-1).
We have reported a method8 for mannose oligosaccha-
ride syntheses using unprotected or lightly protected
sugars as the glycosyl acceptors, and a variety of com-
plex oligosaccharides have been synthesized efficiently.9
In the present research, a concise synthesis of the pen-
tasaccharide repeating unit of the polysaccharide of R.
equi LAM was achieved as shown in Scheme 1. Allyl 3-
Decasaccharide 31, the dimer of the pentasaccharide
repeating unit, was obtained in an alternative way.
Thus, selective 6-O-glycosylation of 4 with the disac-
charide donor 10 gave the trisaccharide 21 (84.5%).
Benzoylation of 21 produced 22, and its 1H NMR
spectrum showed a newly emerged signal at d 5.92 ppm
with J3;4 ¼ J4;5 ¼ 9:9 Hz for H-4 compared to 21, con-
firming the selective 6-O-glycosylation. Deallylation of
22, followed by trichloroacetimidate formation, gave the
trisaccharide donor 24 (74.7% for two steps). Sub-
sequent selective coupling of the monosaccharide
acceptor 3 with 24 yielded the tetrasaccharide 25
(80.1%), and subsequent benzoylation gave the tetra-
saccharide 26 with 2-, 200-chloroacetyl, and 20-, 2000-acetyl
groups. The structure of 26 could allow different sub-
stitution at 2-, 200-, and 20-, 2000-positions. Thus, dechlo-
roacetylation of 26 with thiourea produced
tetrasaccharide acceptor 27 (82.3%) with 2- and 200-free
hydroxyl groups, and subsequent condensation with
perbenzoylated mannosyl donor 11 gave the hexasac-
charide 28 in high yield (85.9%). Then, selective de-
acetylation under the same conditions as those used for
deacetylation of 12 afforded the hexasaccharide acceptor
29 (76.8%) with 20- and 2000-free hydroxyl groups. Fi-
nally, condensation of 29 with 18 (66.7%), followed by
deacylation, gave the target decasaccharide 31 (92.5%).
O-benzoyl-4,6-O-benzylidene-a-D-mannopyranoside (2)
was chosen as the starting material that was prepared in
high yield (90.7%) by selective 3-O-benzoylation of allyl
4,6-O-benzylidene-a-D-mannopyranoside (1) with ben-
zoyl chloride in dichloromethane–pyridine. The 1H
NMR spectrum of 2 showed a characteristic doublet of
doublets at d 5.56 with J2;3 ¼ 3:2 Hz, J3;4 ¼ 10:3 Hz for
H-3, confirming the selectivity. Chloroacetylation of 2,
followed by debenzylidenation, afforded the monosac-
charide acceptor 3, while acetylation followed by de-
benzylidenation afforded the monosaccharide acceptor
4. Subsequent coupling of 3 with 2-O-acetyl-3,4,6-tri-O-
benzoyl-a-D-mannopyranosyl trichloroacetimidate (5)
selectively gave the (1 ! 6)-linked disaccharide
6
(86.4%), and the regioselectivity of the coupling was
confirmed by benzoylation of 6 to give 7 that showed in
its 1H NMR spectrum a newly emerged signal at d
5.93 ppm with J3;4 ¼ J4;5 ¼ 10:1 Hz for H-4 compared to
6. Dechloroacetylation of 7 afforded the disaccharide
10
acceptor 8, while deallylation with PdCl2 followed by
trichloroacetimidate formation,11 yielded the disacchar-
ide donor 10. Condensation of 8 with perbenzoylated
mannosyl trichloroacetimidate 11 produced the trisac-
charide 12 (87.8%), and subsequent selective deacetyl-
ation12 with acetyl chloride in dichloromethane–methanol
(1:5:25 v/v/v) yielded the trisaccharide acceptor 13 in
good yield (78.5%). The other disaccharide block 18 was
obtained through condensation of allyl 3,4,6-tri-O-ben-
1
The H and 13C NMR spectra of 31 showed all of the
characteristic signals such as at d 5.09 (s, 1H, Manp H-
1), 5.08 (s, 1H, Manp H-1), 5.07 (s, 2H, Araf 2H-1), 5.02
(s, 1H, Manp H-1), 5.01 (s, 1H, Manp H-1), 4.99 (s, 2H,
Manp 2H-1), 4.93 (s, 1H, Manp H-1), 4.91 (s, 1H, Manp
H-1); 109.36 (2C, Araf 2C-1), 102.32, 102.19, 101.32,
101.30, 98.17, 98.03, and 97.53 (8C, Manp 8C-1)
(Scheme 2).
zoyl-a-
D
D
-mannopyranoside (15) with perbenzoylated a-
-arabinofuranosyl trichloroacetimidate 1413 to afford
the disaccharide 16 (87.2%). Deallylation and sub-
sequent trichloroacetimidate formation then produced
the disaccharide donor 18 (77.7% for two steps). Finally,
condensation of 18 with the 13 gave pentasaccharide 19
(81.5%), and deacylation in ammonia–saturated meth-
In summary, a facile synthesis of complex arabino-
mannosyl oligosaccharides was achieved through a re-
gio- and stereoselective manner with readily accessible
materials. The described method is suitable for prepa-
ration of a 2,6-branched comb-like mannan with arab-
inofuranose side chains.
1
anol yielded the pentasaccharide 20 (91%). The H and

Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
1763
HO
OCA
O
HO
BzO
b,c
d,c
Ph
O
OH
Ph
O
OH
O
O
O
OAll
a
3
O
HO
BzO
HO
OAc
O
OAll
1
2
OAll
HO
BzO
OAll
4
BzO
OAc
6
7
8
9
R = H, R1 = CA, R2 = All
R = Bz, R1 = CA, R2 = All
R = Bz, R1 = H, R2 = All
R = Bz, R1 = CA, R2 = H
O
BzO
BzO
BzO
f
OAc
O
e
BzO
BzO
g
+
3
h
OR1
O
O
OCNHCCl3
RO
BzO
i
5
10 R = Bz, R1 = CA, R2 = CNHCCl3
OR2
BzO
BzO
BzO
BzO
OBz
O
OR
BzO
OBz
O
BzO
BzO
O
e
BzO
12 R = Ac
13 R = H
+
8
j
BzO
O
O
OCNHCCl3
O
11
BzO
BzO
OAll
BzO
BzO
O
O
BzO
OH
16 R = All
BzO
O
e
h
+
BzO
BzO
OBz
17 R = H
BzO
i
BzO
OCNHCCl3
O
O
18 R = CNHCCl3
OAll
OBz
BzO
BzO
14
15
OR
OR
RO
RO
RO
RO
O
OR
O
O
RO
O
RO
RO
19 R = Bz
20 R = H
e
k
13
+
18
O
RO
OR
O
O
RO
RO
O
O
RO
RO
OAll
Scheme 1. Reagents and conditions: (a) BzCl, CH2Cl2, pyridine, 90.7%; (b), (c) CH2ClCOCl, CH2Cl2, pyridine; 90% TFA, rt, 2 h; 78.5% for two
steps; (d), (c) Ac2O–pyridine; 90% TFA, rt, 2 h; 82.3% for two steps; (e) TMSOTf (0.01–0.05 equiv), CH2Cl2, À20 to 0 °C, 2–4 h, 86.4% for 6, 87.8%
for 12, 87.2% for 16, 81.5% for 19, 84.5% for 21, 80.1% for 25, 85.9% for 28, and 66.7% for 30, respectively; (f) BzCl–pyridine, 91.6% for 7, 89.2% for
22, 85.5% for 26; (g) (NH2)2CS, CH2Cl2–CH3OH, reflux, 16 h, 85.6% for 8, 82.3% for 27; (h) PdCl2, CH3OH, rt, 4 h, 84.5% for 9, 82.4% for 17, 82.7%
for 23; (i) CCl3CN, DBU, CH2Cl2, 2h, 88.7% for 10, 94.3% for 18, 90.3% for 24; (j) CH3OH–2–6% CH3COCl, rt, 12 h, 78.5% for 13, 76.8% for 29; (k)
satd NH3–CH3OH, rt, 96 h, 91.0% for 20, 92.5% for 31.
3. Experimental
3.1. General methods
Bruker ARX 400 spectrometers (400 MHz for 1H,
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 measured using
MALDITOF-MS with CCA as matrix or recorded
with a VG PLATFORM mass spectrometer using the
ESI mode. Thin-layer chromatography (TLC) was
Optical rotations were determined at 25 °C with a
Perkin–Elmer Model 241-Mc automatic polarimeter.
1H NMR and 13C NMR spectra were recorded with

1764
Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
BzO
OAc
O
BzO
BzO
21 R = H, R1 = All
22 R = Bz, R1 = All
23 R = Bz, R1 = H
f
e
O
+
10
4
OCA
O
h
i
BzO
BzO
24 R = Bz, R1 = CNHCCl3
O
OAc
O
RO
BzO
OR1
BzO
OAc
O
BzO
BzO
OR1
O
25 R = H, R1 = CA
26 R = Bz, R1 = CA
27 R = Bz, R1 = H
O
BzO
BzO
f
e
g
+
24
3
O
OAc
O
BzO
BzO
OR1
O
O
RO
BzO
OAll
BzO
BzO
BzO
BzO
OBz
OR
O
O
BzO
BzO
28 R = Ac
29 R = H
j
O
O
O
BzO
BzO
BzO
BzO
BzO
e
+
11
27
O
OBz
O
OR
O
BzO
BzO
O
O
O
BzO
BzO
OAll
OR
RO
RO
RO
RO
O
OR
O
O
RO
O
RO
RO
OR
O
RO
OR
O
RO
RO
O
RO
RO
RO
RO
O
O
OR
O
30 R = Bz
31 R = H
O
k
RO
e
O
+
18
29
RO
RO
O
RO
RO
O
O
OR
O
O
RO
RO
O
O
RO
RO
OAll
Scheme 2. Reagents and conditions: (a) BzCl, CH2Cl2, pyridine, 90.7%; (b), (c) CH2ClCOCl, CH2Cl2, pyridine; 90% TFA, rt, 2 h; 78.5% for two
steps; (d), (c) Ac2O–pyridine; 90% TFA, rt, 2 h; 82.3% for two steps; (e) TMSOTf (0.01–0.05equiv), CH2Cl2, À20 to 0 °C, 2–4 h, 86.4% for 6, 87.8%
for 12, 87.2% for 16, 81.5% for 19, 84.5% for 21, 80.1% for 25, 85.9% for 28, and 66.7% for 30, respectively; (f) BzCl–pyridine, 91.6% for 7, 89.2% for
22, 85.5% for 26; (g) (NH2)2CS, CH2Cl2–CH3OH, reflux, 16 h, 85.6% for 8, 82.3% for 27; (h) PdCl2, CH3OH, rt, 4 h, 84.5% for 9, 82.4% for 17, 82.7%
for 23; (i) CCl3CN, DBU, CH2Cl2, 2h, 88.7% for 10, 94.3% for 18, 90.3% for 24; (j) CH3OH–2–6% CH3COCl, rt, 12 h, 78.5% for 13, 76.8% for 29; (k)
satd NH3–CH3OH, rt, 96 h, 91.0% for 20, 92.5% for 31.
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 Â 240 mm,

Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
1765
18 Â 300 mm, 35 Â 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.
end of which time TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The mixture
was diluted with toluene (80 mL) and concentrated in
vacuo directly. The residue was passed through a short
silica gel column with 1:1 petroleum ether–EtOAc as the
eluent to give 3 (0.94 g, 78.5% for two steps) as syrup:
3.2. General procedure for the glycosylations
½aŠ þ96.7 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
A mixture of the donor and acceptor was dried together
under high vacuum for 2 h, then dissolved in anhyd
CH2Cl2 (1 mmol donor in 20 mL). TMSOTf (0.05 equiv)
was added dropwise at À20 °C with nitrogen protection.
The reaction mixture was stirred for 3 h, during which
time the temperature was gradually raised to ambient
temperature. Then the mixture was neutralized with
Et3N. Concentration of the reaction mixture, followed
by purification on a silica gel column, gave the desired
products.
d 8.00–7.43 (m, 5H, Ph), 5.96–5.86 (m, 1H, –CH2–
CH@CH2), 5.52 (dd, 1H, J2;3 ¼ 3:3 Hz, J3;4 ¼ 9:9 Hz, H-
3), 5.44 (dd, 1H, J1;2 ¼ 1:6 Hz, J2;3 ¼ 3:3 Hz, H-2), 5.36-
5.24 (m, 2H, –CH2–CH@CH2), 4.93 (d, 1H, J1;2
¼
1:6 Hz, H-1), 4.26–4.02 (m, 5H, H-4, CH2ClCO, –CH2–
CH@CH2), 3.93–3.82 (m, 3H, H-5, H-6). Anal. Calcd
for C18H21ClO8: C 53.94; H 5.28. Found: C 54.09; H
5.33.
3.5. Allyl 2-O-acetyl-3-O-benzoyl-a-D-mannopyranoside
(4)
3.3. Allyl 3-O-benzoyl-4,6-di-O-benzylidene-a-D-manno-
pyranoside (2)
To a solution of 16 (412 mg, 1 mmol) in pyridine (10 mL)
was added Ac2O (5 mL, 5 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,
the residue was dissolved in 90% TFA (10 mL), and the
mixture was stirred for 2 h at rt, at the end of which time
TLC (2:1 petroleum ether–EtOAc) indicated that the
reaction was complete. The mixture was diluted with
toluene (40 mL) and concentrated in vacuo directly. The
residue was passed through a short silica gel column
with 1:1 petroleum ether–EtOAc as the eluent to give 4
Compound 1 (1.54 g, 5.0 mmol) was dissolved in dry
CH2Cl2 (20 mL) containing pyridine (4 mL), then under
N2 protection, benzoyl chloride (0.6 mL, 5.1 mmol) in
anhyd CH2Cl2 (5 mL) was added dropwise to the solu-
tion within 30 min at 0 °C. The reaction mixture was
slowly raised to room temperature (rt) and stirred for
2 h, at the end of which time TLC (3:1 petroleum ether–
EtOAc) indicated that the reaction was complete. The
reaction mixture was diluted with CH2Cl2 (50 mL),
washed with water, 1 N HCl, and dried over Na2SO4.
The solution was concentrated, and purification of the
residue by column chromatography on a silica gel col-
umn (4:1 petroleum ether–EtOAc) gave compound 2
(302 mg, 82.3% for two steps) as syrup: ½aŠ þ62.6 (c 1.0,
D
CHCl3); 1H NMR (400 MHz, CDCl3): d 8.00–7.43
(m, 5H, Ph), 5.96–5.86 (m, 1H, –CH2–CH@CH2), 5.48
(dd, 1H, J2;3 ¼ 3:5 Hz, J3;4 ¼ 9:9 Hz, H-3), 5.39 (dd, 1H,
J1;2 ¼ 1:6 Hz, J2;3 ¼ 3:5 Hz, H-2), 5.35–5.23 (m, 2H,
–CH2–CH@CH2), 4.90 (d, 1H, J1;2 ¼ 1:6 Hz, H-1), 4.25–
4.01 (m, 3H, H-4, –CH2–CH@CH2), 3.94–3.82
(m, 3H, H-5, H-6), 2.15 (s, 3H, CH3CO). Anal.
Calcd for C18H22O8: C 59.01; H 6.05. Found: C 59.28; H
6.11.
1
(1.87 g, 90.7%) as a syrup: ½aŠ À8.6 (c 1.0, CHCl3); H
D
NMR (400 MHz, CDCl3): d 8.05–7.28 (m, 10H, 2Ph),
5.95–5.86 (m, 1H, –CH2–CH@CH2), 5.57 (s, 1H,
PhCH), 5.56 (dd, 1H, J2;3 ¼ 3:2 Hz, J3;4 ¼ 10:3 Hz, H-3),
5.34–5.20 (m, 2H, –CH2–CH@CH2), 4.89 (d, 1H,
J1;2 ¼ 1:2 Hz, H-1), 4.30-3.87 (m, 7H, H-2, H-4, H-5, H-
6, –CH2–CH@CH2). Anal. Calcd for C23H24O7: C 66.98;
H 5.87. Found: C 67.21; H 5.92.
3.6. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
D
-mannopyr-
-manno-
3.4. Allyl 3-O-benzoyl-2-O-chloroacetyl-a-
anoside (3)
D
-mannopyr-
anosyl-(1 ! 6)-3-O-benzoyl-2-O-chloroacetyl-a-
D
pyranoside (6)
Compound 2 (1.24 g, 3.0 mmol) was dissolved in dry
CH2Cl2 (20 mL) containing pyridine (3 mL), then under
N2 protection, chloroacetyl chloride (0.27 mL,
3.3 mmol) in anhyd CH2Cl2 (5 mL) was added dropwise
to the solution. The reaction mixture was stirred for 2 h,
then was diluted with CH2Cl2 (40 mL), washed with
water, 1 N HCl, and dried over Na2SO4. The solution
was concentrated, the residue was dissolved in 90% TFA
(20 mL), and the mixture was stirred for 2 h at rt, at the
Donor 5 (996 mg, 1.47 mmol) was coupled with acceptor
3 (490 mg, 1.22 mmol) as described in the general pro-
cedure, and the product was purified by chromatogra-
phy with 3:1 petroleum ether–EtOAc as the eluent to
give 10 (969 mg, 86.4%) as a foamy solid: ½aŠ þ49.6 (c
D
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): d 8.07–7.26
(m, 20H, 4Ph), 5.98–5.88 (m, 2H, H-40, –CH2–
0
0
0
0
CH@CH2), 5.84 (dd, 1H, J2 ;3 ¼ 3:3 Hz, J3 ;4 ¼ 10:1 Hz,
H-30), 5.60 (dd, 1H, J1 ;2 ¼ 1:8 Hz, J2 ;3 ¼ 3:3 Hz, H-20),
0
0
0
0

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Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
5.55 (dd, 1H) 5.38–5.24 (m, 2H, –CH2–CH@CH2), 5.15
5.87 (dd, 1H, J3;4 ¼ J4;5 ¼ 10:1 Hz, H-4), 5.85 (dd, 1H,
0 0 0 0
(d, 1H, J1 ;2 ¼ 1:8 Hz, H-10), 4.95 (d, 1H, J1;2 ¼ 1:8 Hz,
H-1), 4.66–4.61 (m, 1H, H-50), 4.54–4.48 (m, 2H, H-5,
H-60a), 4.36 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, H-4), 4.25
(ABq, 2H, J ¼ 20:4 Hz, CH2ClCO), 4.24–3.90 (m, 5H,
H-6a, H-6b, H-60b, –CH2–CH@CH2), 2.11 (s, 3H,
CH3CO). Anal. Calcd for C47H45ClO17: C 61.54; H 4.94.
Found: C 61.38; H 5.01.
J2 ;3 ¼ 3:2 Hz, J3 ;4 ¼ 10:1 Hz, H-30), 5.70 (dd, 1H,
J2;3 ¼ 3:1 Hz, J3;4 ¼ 10:1 Hz, H-3), 5.51 (dd, 1H,
0
0
J1 ;2 ¼ 1:5 Hz, J2 ;3 ¼ 3:2 Hz, H-20), 5.47–5.29 (m, 2 H,
0
0
0
0
–CH2–CH@CH2), 5.04 (d, 1H, J1 ;2 ¼ 1:5 Hz, H-10),
5.03 (d, 1H, J1;2 ¼ 1:6 Hz, H-1), 4.50–3.71 (m, 9H, H-2,
H-5, H-50, H-6, H-60, –CH2–CH@CH2), 2.12 (s, 3H,
CH3CO). Anal. Calcd for C52H48O17: C 66.10; H 5.12.
Found: C 65.87; H 5.09.
0
0
3.7. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyr-
anosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
D
-
3.9. 2-O-Acetyl-3,4,6-tri-O-benzoyl-a-D-mannopyranos-
mannopyranoside (7)
yl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
D-
mannopyranosyl trichloroacetimidate (10)
To a solution of 6 (842 mg, 0.92 mmol) in pyridine
(10 mL) was added benzoyl chloride (0.13 mL,
1.1 mmol). After stirring the mixture overnight at rt,
TLC (2:1 petroleum ether–EtOAc) indicated that the
reaction was complete. MeOH (one drop) was added to
the reaction mixture, and stirring was continued for
10 min. Water (10 mL) was added, and the mixture was
extracted with CH2Cl2 (3 Â 10 mL). The extract was
washed with M HCl and satd aq NaHCO3, dried (Na2
SO4), and concentrated. Purification by flash chroma-
tography (3:1 petroleum ether–EtOAc) gave 7 (859 mg,
To a solution of 7 (515 mg, 0.50 mmol) in anhyd MeOH
(10 mL) was added PdCl2 (30 mg). After stirring the
mixture at rt for 2 h, TLC (2:1 petroleum ether–EtOAc)
indicated that the reaction was complete. The mixture
was filtered, the solution was concentrated to dryness,
and the resultant residue was purified by flash chroma-
tography (2.5:1 petroleum ether–EtOAc) to give 9
(418 mg, 84.5%) as a white foam. A mixture of 9
(418 mg, 0.43 mmol), trichloroacetonitrile (80 lL,
0.80 mmol) and 1,8-diazabicyclo[5.4.0]-undecene (DBU)
(25 lL) in dry CH2Cl2 (10 mL) was stirred under nitro-
gen for 3 h and then concentrated. The residue was
purified by flash chromatography (3:1 petroleum ether–
91.6%) as a foamy solid: ½aŠ þ40.56 (c 1.0, CHCl3); 1H
D
NMR (400 MHz, CDCl3): 8.04–7.26 (m, 25H, 5Ph),
6.05–5.95 (m, 1H, –CH2–CH@CH2), 5.97 (dd, 1H,
J3 ;4 ¼ J4 ;5 ¼ 10:1 Hz, H-40), 5.93 (dd, 1H, J3;4
¼
EtOAc) to give 10 (425 mg, 88.7%) as a foamy solid: ½aŠ
0
0
0
0
D
1
0
0
J4;5 ¼ 10:1 Hz, H-4), 5.88 (dd, 1H, J2 ;3 ¼ 3:3 Hz,
þ55.5 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
J3 ;4 ¼ 10:1 Hz, H-30), 5.85 (dd, 1H, J2;3 ¼ 3:3 Hz,
8.97 (s, 1H, CNHCCl3), 8.08–7.26 (m, 25H, 5Ph), 6.44
0
0
0
0
0
0
J3;4 ¼ 10:1 Hz, H-3), 5.58 (dd, 1H, J1 ;2 ¼ 1:8 Hz,
(d, 1H, J1;2 ¼ 1:5 Hz, H-1), 6.12 (dd, 1H, J3 ;4
¼
J2 ;3 ¼ 3:3 Hz, H-20), 5.55 (dd, 1H, J1;2 ¼ 1:8 Hz,
J4 ;5 ¼ 10:0 Hz, H-40), 5.93 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:9 Hz,
0
0
0
0
J2;3 ¼ 3:3 Hz, H-2), 5.47–5.31 (m, 2H, –CH2–CH@CH2),
H-4), 5.91–5.82 (m, 2H, H-3, H-30), 5.78 (dd, 1H,
5.04 (d, 1H, J1 ;2 ¼ 1:8 Hz, H-10), 5.03 (d, 1H,
J1 ;2 ¼ 1:4 Hz, J2 ;3 ¼ 3:2 Hz, H-20), 5.57 (dd, 1H,
0
0
0
0
0
0
0
0
J1;2 ¼ 1:8 Hz, H-1), 4.47 (dd, 1H, J5 ;6 a ¼ 2:4 Hz,
J1;2 ¼ 1:5 Hz, J2;3 ¼ 3:0 Hz, H-2), 5.01 (d, 1H,
J6 a;6 b ¼ 11:6 Hz, H-60a), 4.43–3.72 (m, 9H, H-5, H-50,
H-6a, H-6b, H-60b, CH2ClCO, –CH2–CH@CH2), 2.11
(s, 3H, CH3CO). Anal. Calcd for C54H49ClO18: C 63.50;
H 4.84. Found: C 63.75; H 4.78.
J1 ;2 ¼ 1:4 Hz, H-10), 4.52–3.78 (m, 8H, H-5, H-50, H-6,
H-60, CH2ClCO), 2.12 (s, 3H, CH3CO). Anal. Calcd for
C53H45Cl4NO18: C 56.55; H 4.03. Found: C 56.32; H
3.97.
0
0
0
0
3.8. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
D
-mannopyr-
-mannopyranoside
3.10. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
D
-manno-
-manno-
D-mannopyrano-
anosyl-(1 ! 6)-3,4-di-O-benzoyl-a-
D
pyranosyl-(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a-
D
(8)
pyranosyl-(1 ! 2)]-3,4-di-O-benzoyl-a-
side (12)
To a solution of 7 (320 mg, 0.31 mmol) in MeOH
(10 mL)–CH2Cl2 (15 mL) was added thiourea (600 mg),
and the mixture was refluxed for 16 h, at the end of
which time TLC (2:1 petroleum ether–EtOAc) indicated
that the reaction was complete. The mixture was con-
centrated. The residue was passed through a silica-gel
column with 3:1 petroleum ether–EtOAc as the eluent to
give 8 (253 mg, 85.6% for two steps) as a foamy solid:
Compound 8 (240 mg, 0.25 mmol) and 11 (226 mg,
0.31 mmol) were coupled under the same conditions as
those used for preparation of 6 from 3 and 5, giving 12
(340 mg, 87.8%) as a foamy solid: ½aŠ À32.5 (c 1.0,
D
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.13–7.05 (m,
45H, 9Ph), 6.17 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, H-4), 6.10–
5.91 (m, 7H, H-2, 3H-3, 2H-4, –CH2–CH@CH2), 5.63
(dd, 1H, J1;2 ¼ 1:4 Hz, J2;3 ¼ 2:9 Hz, H-2), 5.50–5.29 (m,
2H, –CH2–C@HCH2), 5.32 (d, 1H, J1;2 ¼ 1:2 Hz, H-1),
5.27 (d, 1H, J1;2 ¼ 1:3 Hz, H-1), 5.27 (d, 1H,
½aŠ þ32.2 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
d 8.01–7.26 (m, 25H, 5Ph), 6.08–5.98 (m, 1H, –CH2–
CH@CH2), 5.88 (dd, 1H, J3 ;4 ¼ J4 ;5 ¼ 10:1 Hz, H-40),
0
0
0
0

Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
1767
J1;2 ¼ 1:4 Hz, H-1), 5.08 (d, 1H, J1;2 ¼ 1:5 Hz, H-1),
3.13. 2,3,5-Tri-O-benzoyl-a-
D
-arabinofuranosyl-(1 ! 2)-
4.77–3.79 (m, 12H, H-2, 3H-5, 6H-6, –CH2–CH@CH2),
2.06 (s, 3H, CH3CO); 13C NMR (100 MHz, CDCl3): d
169.67 (CH3CO), 166.09, 165.99, 165.87, 165.67, 165.60,
165.29, 164.95, 164.81, 167.80 (PhCO), 99.66, 97.86,
97.55 (C-1), 71.06, 70.23, 70.03, 69.97, 69.76, 69.74,
68.83, 68.67, 67.35, 66.86, 66.83, 66.73, 63.02, 62.89,
60.34, 20.69. Anal. Calcd for C86H74O26: C 67.80; H
4.90. Found: C 67.68; H 4.83.
3,4,6-tri-O-benzoyl-a-D-mannopyranosyl trichloroacet-
imidate (18)
Deallylation of disaccharide 16 (415 mg, 0.07 mmol),
followed by trichloroacetimidation under the same
conditions as those used for preparation of 10 from 7
gave a residue that was purified by flash chromatogra-
phy (3:1 petroleum ether–EtOAc) to give 18 (357 mg,
77.7% for two steps) as a foamy solid: ½aŠ À14.5 (c 1.0,
D
1
3.11. Allyl 3,4,6-tri-O-benzoyl-a-
(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a-
(1 ! 2)]-3,4-di-O-benzoyl-a- -mannopyranoside (13)
D
-mannopyranosyl-
CHCl3); H NMR (400 MHz, CDCl3): d 8.81 (s, 1H,
CNHCCl3), 8.11–7.23 (m, 30H, 6Ph), 6.56 (d, 1H,
D
-mannopyranosyl-
D
J1;2 ¼ 1:6 Hz, Manp H-1), 6.15 (dd, 1H, J3;4 ¼
J4;5 ¼ 10:1 Hz, Manp H-4), 5.89 (dd, 1H, J2;3 ¼ 3:2 Hz,
J3;4 ¼ 10:1 Hz, Manp H-3), 5.72 (d, 1H, J2;3 ¼ 1:1 Hz,
Araf H-2), 5.63 (dd, 1H, J2;3 ¼ 1:1 Hz, J3;4 ¼ 4:4 Hz,
Araf H-3), 5.53 (s, 1H, Araf H-1), 4.81–4.37 (m, 7H,
Manp H-2, H-5, 2 H-6, Araf H-4, 2H-5). Anal. Calcd for
C55H44Cl3NO16: C 61.09; H 4.10. Found: C 61.18; H
4.04.
To a solution of 12 (325 mg, 0.21 mmol) in anhyd
CH2Cl2 (5 mL) was added anhyd MeOH (25 mL), then
acetyl chloride (1 mL) was added to the reaction mixture
at 0 °C. The solution was stoppered in a flask and stirred
at rt until TLC (1:1 petroleum ether–EtOAc) showed
that the reaction was complete. The solution was neu-
tralized with Et3N, then concentrated to dryness. The
residue was passed through a short silica gel column to
3.14. Allyl [2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl-
give 13 (248 mg, 78.5%) as a foamy solid: ½aŠ À11.6 (c
(1 ! 2)-3,4,6-tri-O-benzoyl-a-
D
-mannopyranosyl-
-mannopyranosyl-(1 ! 6)-
D
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): d 8.02–7.04
(1! 2)]-3,4,6-tri-O-benzoyl-a-
D
(m, 45H, 9Ph), 6.25 (dd, 1H, J3;4 ¼ J4;5 ¼ 9:8 Hz, H-4),
6.11–5.79 (m, 7H, H-2, 3H-3, 2H-4, –CH2–CH@CH2),
5.54 (s, 1H, H-1), 5.40–5.27 (m, 2H, –CH2–CH@CH2),
5.37 (s, 1H, H-1), 5.17 (s, 1H, H-1), 4.81 (m, 1H, H-2),
4.72–3.73 (m, 12H, H-2, 3H-5, 6H-6, –CH2–CH@CH2);
13C NMR (100 MHz, CDCl3): d 166.25, 166.10, 165.83,
165.75, 165.65, 165.64, 165.33, 164.79, 164.71 (PhCO),
100.60, 99.41, 97.97 (C-1), 72.80, 71.34, 71.13, 70.35,
69.89, 69.70, 68.89, 68.63, 67.34, 67.10, 66.54, 64.65,
63.74, 63.01, 60.39. Anal. Calcd for C84H72O25: C 68.10;
H 4.90. Found: C 67.95; H 4.91.
[2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl-(12)]-3,4-
di-O-benzoyl-a-D-mannopyranoside (19)
As described in the general procedure, 13 (235 mg,
0.16 mmol) and 18 (206 mg, 0.19 mmol) were coupled,
and the product was purified by silica gel column
chromatography with 1.5:1 petroleum ether–EtOAc as
the eluent to give 19 (310 mg, 81.5%) as a foamy solid:
½aŠ À34.7 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
d 8.09–6.95 (m, 75H, 15Ph), 6.14–5.83 (m, 10H, Manp
H-2, 4H-3, 4H-4, –CH2–CH@CH2), 5.54 (s, 1H, Araf H-
2), 5.53 (d, 1H, J3;4 ¼ 4:3 Hz, Araf H-3), 5.45–5.28 (m,
2H, –CH2–CH@CH2), 5.32 (s, 1H, Araf H-1), 5.28 (s,
1H, Manp H-1), 5.22 (s, 2H, Manp 2H-1), 5.20 (s, H,
Manp H-1), 4.75–3.64 (m, 20H, Manp 3H-2, 4H-5, 8H-
6, Araf H-4, 2H-5, –CH2–CH@CH2); 13C NMR
(100 MHz, CDCl3): d 166.19, 166.07, 165.98, 165.86,
165.66, 165.65, 165.57, 165.54, 165.34, 165.31, 165.28,
165.23, 164.85, 164.83, 164.78 (PhCO), 106.87 (Araf C-
1), 100.86, 99.46, 98.63, 97.98 (C-1), 81.90, 81.77, 76.26,
75.45, 71.74, 71.26, 70.90, 70.19, 69.80, 69.74, 69.64,
68.68, 68.65, 67.59, 67.54, 66.91, 66.73, 63.77, 63.51,
63.11, 62.97. Anal. Calcd for C137H114O40: C 68.55; H
4.79. Found: C 68.37; H 5.70.
3.12. Allyl 2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl-
(1 ! 2)-3,4,6-tri-O-benzoyl-a- -mannopyranoside (16)
D
Acceptor 15 (266 mg, 0.50 mmol) was coupled with do-
nor 14 (364 mg, 0.60 mmol) as described in the general
procedure, and the product was purified by chroma-
tography with 1.5:1 petroleum ether–EtOAc as the elu-
ent to give 16 (426 mg, 87.2%) as a foamy solid: ½aŠ
D
1
À36.6 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
8.14–7.21
(m,
30H,
6Ph),
6.01
(dd,
1H,
J3;4 ¼ J4;5 ¼ 10:0 Hz, Manp H-4), 6.10–5.90 (m, 1H,
–CH2–CH@CH2), 5.87 (dd, 1H, J2;3 ¼ 3:2 Hz,
J3;4 ¼ 10:0 Hz, Manp H-3), 5.71 (d, 1H, J2;3 ¼ 1:1 Hz,
Araf H-2), 5.62 (dd, 1H, J2;3 ¼ 1:1 Hz, J3;4 ¼ 3:5 Hz,
Araf H-3), 5.48 (s, 1H, Araf H-1), 5.33–5.18 (m, 2H,
–CH2–CH@CH2), 5.13 (d, 1H, J1;2 ¼ 1:7 Hz, Manp
H-1), 4.76–4.36 (m, 7H, Manp H-2, H-5, 2H-6, Araf
H-4, 2H-5), 4.31–4.06 (m, 2H, –CH2–CH@CH2). Anal.
Calcd for C56H48O16: C 68.85; H 4.95. Found: C 68.95;
H 5.02.
3.15. Allyl [a-
anosyl-(1 ! 2)]-a-
pyranosyl-(1 ! 2)]-a-
D
-arabinofuranosyl-(1 ! 2)-a-
-mannopyranosyl-(1 ! 6)-[a-
-mannopyranoside (20)
D
-mannopyr-
D
D
-manno-
D
Pentasaccharide 19 (299 mg, 0.13 mmol) was dissolved in
satd NH3–MeOH (60 mL). After 96 h at rt, the reaction
mixture was concentrated, and the residue was purified

1768
Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
0
0
0
0
by chromatography on Sephadex LH-20 (MeOH) to
afford 30 (95 mg, 91.0%) as a foamy solid: ½aŠ þ78.5 (c
1H, J3 ;4 ¼ J4 ;5 ¼ 10:2 Hz, H-40) 5.92 (dd, 1H,
00 ;300
J3;4 ¼ J4;5 ¼ 9:9 Hz, H-4), 5.91 (dd, 1H, J2
¼ 3:0 Hz,
D
1
00 ;400
J3
0
0
1.0, H2O); H NMR (400 MHz, D2O): d 5.89–5.79 (m,
¼ 10:9 Hz, H-300), 5.85 (dd, 1H, J2 ;3 ¼ 3:0 Hz,
0
0
1H, –CH2–CH@CH2), 5.24 (d, 1H, J ¼ 17:2 Hz, –CH2–
CH@CHtrans), 5.16 (d, 1H, J ¼ 10:4 Hz, –CH2–
CH@CHcis), 5.06 (s, 1H, Manp H-1), 5.05 (s, 1H, Araf
H-1), 5.01 (s, 1H, Manp H-1), 4.99 (s, 1H, Manp H-1),
4.89 (s, 1H, Manp H-1); 13C NMR (100 MHz, D2O): d
109.36 (Araf C-1), 102.32, 101.36, 97.95, 97.47 (Manp C-
1), 83.62, 81.19, 78.85, 78.67, 77.43, 76.44, 73.31, 73.15,
72.82, 71.07, 70.47, 70.38, 70.08, 70.04, 68.42, 66.98,
66.81, 66.75, 66.69, 65.66, 61.15, 61.06, 60.91. Anal.
Calcd for C32H54O25: C 45.82; H 6.49. Found: C 46.01;
H 6.55.
J3 ;4 ¼ 10:2 Hz, H-30), 5.84 (dd, 1H, J2;3 ¼ 3:1 Hz,
00 ;200
J3;4 ¼ 9:9 Hz, H-3), 5.65 (dd, 1H, J1
¼ 1:2 Hz,
00 ;300
0
0
J2
¼ 3:0 Hz, H-2), 5.54 (dd, 1H, J1 ;2 ¼ 1:2 Hz,
0
0
J2 ;3 ¼ 3:0 Hz, H-2), 5.48 (dd, 1H, J1;2 ¼ 1:3 Hz,
J2;3 ¼ 3:1 Hz, H-2), 5.45–5.27 (m,
2
H, –CH2–
00 ;200
CH@CH2), 5.07 (d, 1H, J1
¼ 1:2 Hz, H-100), 5.05 (d,
1H, J1 ;2 ¼ 1:2 Hz, H-10), 4.88 (d, 1H, J1;2 ¼ 1:3 Hz, H-
1), 4.46–3.49 (m, 13H, 3H-5, 6H-6, –CH2–CH@CH2,
CH2ClCO), 2.20 (s, 3H, CH3CO), 2.11 (s, 3H, CH3CO);
0
0
13C NMR (100 MHz, CDCl3):
d 170.26, 169.65
(CH3CO), 166.78, 165.96, 165.48, 165.43, 165.33,
165.28, 165.18 (PhCO), 97.55, 97.37, 96.73 (C-1), 71.34,
70.19, 69.90, 69.89, 69.79, 69.69, 69.68, 68.86, 68.75,
66.67, 66.62, 66.51, 66.14, 65.67, 62.92, 40.74, 20.76,
20.68. Anal. Calcd for C76H69ClO26: C 63.66; H 4.85.
Found: C 63.48; H 4.92.
3.16. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
-mannopyranosyl-(1 ! 6)-2-O-acetyl-3-O-benzoyl-a-
mannopyranoside (21)
D-manno-
D
D-
Donor 10 (411 mg, 0.37 mmol) was coupled with
acceptor 4 (160 mg, 0.44 mmol) as described in the
general procedure, and the product was purified by
chromatography with 2:1 petroleum ether–EtOAc as the
3.18. 2-O-Acetyl-3,4,6-tri-O-benzoyl-a-
syl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
mannopyranosyl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-a-
-mannopyranosyl trichloroacetimidate (24)
D-mannopyrano-
D
-
D
eluent to give 21 (410 mg, 84.5%) as a foamy solid: ½aŠ
D
1
þ42.7 (c 0.6, CHCl3); H NMR (400 MHz, CDCl3): d
Deallylation of trisaccharide 22 (365 mg, 0.25 mmol),
followed by trichloroacetimidation under the same
conditions as those used for preparation of 10 from 7,
gave a residue that was purified by flash chromatogra-
phy (3:1 petroleum ether–EtOAc) to give 24 (292 mg,
8.03–7.26 (m, 30H, 6Ph), 5.98–5.88 (m, 1H, –CH2–
00 ;400
00 ;500
CH@CH2), 5.91 (dd, 1H, J3
¼ J4
¼ 10:1 Hz, H-
400), 5.90 (dd, 2H, J2
¼ J2 ;3 ¼ 3:3 Hz, J3
¼ J3 ;4
¼
¼
00 ;300
00 ;400
0
0
0
0
10:1 Hz, H-300, H-300), 5.85 (dd, 1H, J3 ;4 ¼ J4 ;5
0
0
0
0
10:1 Hz, H-40), 5.66 (dd, 1H, J1
¼ 1:6 Hz,
74.7% for two steps) as a foamy solid: ½aŠ þ50.6 (c 1.5,
00 ;200
D
1
00 ;300
J2
¼ 3:3 Hz, H-200), 5.54 (dd, 1H, J2;3 ¼ 3:4 Hz,
CHCl3); H NMR (400 MHz, CDCl3): d 8.94 (s, 1H,
CNHCCl3), 8.05–7.26 (m, 35H, 7Ph), 6.44 (s, 1H, H-1),
0
0
J3;4 ¼ 9:9 Hz, H-3), 5.52 (dd, 1H, J1 ;2 ¼ 1:6 Hz,
J2 ;3 ¼ 3:3 Hz, H-20), 5.44 (dd, 1H, J1;2 ¼ 1:7 Hz,
6.10 (dd, 1H, J3
¼ 10:2 Hz, H-400), 5.96–5.75
00 ;400
00 ;500
¼ J4
0
0
J2;3 ¼ 3:4 Hz, H-2), 5.39–5.21 (m, 2H, –CH2–CH@CH2),
(m, 5H, H-300, H-30, H-3, H-40, H-4), 5.75 (s, 1H, H-200),
5.61 (s, 1H, H-20), 5.37 (s, 1H, H-2), 5.03 (s, 1H, H-100),
4.86 (s, 1H, H-10), 4.56–3.49 (m, 11H, 3H-5, 6H-6,
CH2ClCO), 2.24 (s, 3H, CH3CO), 2.09 (s, 3H, CH3CO).
Anal. Calcd for C75H65Cl4NO26: C 58.57; H 4.26.
Found: C 58.33; H 4.18.
5.17 (d, 1H, J1
¼ 1:6 Hz, H-100), 5.02 (d, 1H,
00 ;200
J1 ;2 ¼ 1:6 Hz, H-10), 4.97 (d, 1H, J1;2 ¼ 1:7 Hz, H-1),
4.52–3.72 (m, 14H, H-4, 3H-5, 6H-6, –CH2–CH@CH2,
CH2ClCO), 2.16 (s, 3H, CH3CO), 2.12 (s, 3H, CH3CO).
Anal. Calcd for C69H65ClO25: C 62.33; H 4.93. Found:
C 62.13; H 4.95.
0
0
3.19. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
-mannopyranosyl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-
a-
-mannopyranosyl-(1 ! 6)-3-O-benzoyl-2-O-chloro-
acetyl-a- -mannopyranoside (25)
D-manno-
3.17. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
-mannopyranosyl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-
a- -mannopyranoside (22)
D-manno-
D
D
D
D
D
Compound 21 (395 mg, 0.3 mmol) was benzoylated un-
der the same conditions as those used for preparation of
7 from 6, giving a residue that was purified by flash
chromatography (2.5:1 petroleum ether–EtOAc) to
Donor 24 (277 mg, 0.18 mmol) was coupled with
acceptor 3 (87 mg, 0.22 mmol) as described in the gen-
eral procedure to give a crude product that was purified
by flash chromatography (1.5:1 petroleum ether–
furnish 22 (380 mg, 89.2%) as a foamy solid: ½aŠ þ44.6
EtOAc) to give 25 (256 mg, 80.1%) as a foamy solid: ½aŠ
D
D
1
1
(c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d 7.98–
7.26 (m, 35H, 7Ph), 6.02–5.83 (m, 7H, 3H-3, 3H-4,
þ50.2 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
000 ;4000
8.00–7.26 (m, 40H, 8Ph), 6.00 (dd, 1H, J3
¼
–CH2–CH@CH2), 6.02–5.91 (m, 1H, –CH2–CH@CH2),
J4
¼ 10:0 Hz, H-4000), 5.96–5.81 (m, 6H, H-3000, H-300,
000 ;5000
5.99 (dd, 1H, J3
¼ J4
¼ 10:9 Hz, H-400), 5.98 (dd,
H-30, H-400, H-40, –CH2–CH@CH2), 5.63 (dd, 1H,
00 ;400
00 ;500

Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
1769
J1
¼ 1:4 Hz, J2
¼ 3:2 Hz, H-2000), 5.58 (dd, 1H,
45H, 9Ph), 6.14–6.06 (m, 2H, H-4000, –CH2–CH@CH2),
5.70–5.96 (m, 7H, 4H-3, 3H-4), 5.55–5.33 (m, 2H, –
CH2–CH@CH2), 5.52–5.50 (m, 2H, H-20, H-2000), 5.21 (d,
000 ;2000
000 ;3000
J2;3 ¼ 3:2 Hz, J3;4 ¼ 10:0 Hz, H-3), 5.57 (dd, 1H,
J1
0
¼ 1:6 Hz, J2
¼ 3:2 Hz, H-200), 5.52 (dd, 1H,
00 ;200
00 ;300
J1 ;2 ¼ 1:6 Hz, J2 ;3 ¼ 3:2 Hz, H-20), 5.49 (dd, 1H,
2H, J1
¼ J1
¼ 1:4 Hz, H-1000, H-100), 5.03 (d, 1H,
000 ;2000
00 ;200
0
0
0
J1 ;2 ¼ 1:5 Hz, H-10), 4.91 (d, 1H, J1;2 ¼ 1:6 Hz, H-1),
4.72–3.50 (m, 16H, 2H-2, 4H-5, 8H-6, –CH2–
CH@CH2), 2.15 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO);
0
0
J1;2 ¼ 1:6 Hz, J2;3 ¼ 3:2 Hz, H-2), 5.39–5.22 (m, 2H,
¼ 1:4 Hz, H-1000),
000 ;2000
–CH2–CH@CH2), 5.18 (d, 1H, J1
5.06 (d, 1H, J1
¼ 1:6 Hz, H-100), 5.00 (d, 1H,
00 ;200
J1 ;2 ¼ 1:6 Hz, H-10), 4.92 (d, 1H, J1;2 ¼ 1:6 Hz, H-1),
4.48–3.59 (m, 19H, H-4, 4H-5, 8H-6, 2CH2ClCO,
–CH2–CH@CH2), 2.17 (s, 3H, CH3CO), 2.07 (s, 3H,
CH3CO). Anal. Calcd for C91H84Cl2O33: C 61.52; H
4.77. Found: C 61.21; H 4.85.
13C NMR (100 MHz, CDCl3):
d 170.09, 169.75
0
0
(CH3CO), 166.04, 166.01, 165.73, 165.64, 165.61,
165.59, 165.58, 165.57, 165.34 (PhCO), 98.80, 98.68,
97.75, 96.74 (C-1), 72.94, 72.58, 70.24, 70.04, 69.97,
69.85, 69.78, 69.49, 69.14, 68.75, 68.72, 68.27, 67.70,
67.07, 66.83, 66.74, 66.41, 66.35, 66.29, 20.80, 20.73.
Anal. Calcd for C94H86O32: C 65.35; H 5.02. Found: C
65.07; H 4.98.
3.20. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloroacetyl-a-
-mannopyranosyl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-
a-
-mannopyranosyl-(1 ! 6)-3,4-di-O-benzoyl-2-O-chloro-
acetyl-a- -mannopyranoside (26)
D-manno-
D
D
3.22. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a-
pyranosyl-(1 ! 2)]-3,4-di-O-benzoyl-a- -mannopyranos-
yl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-a- -mannopyr-
-mannopyr-
-mannopyranoside
D
-manno-
D
D-manno-
D
Compound 25 (242 mg, 0.14 mmol) was benzoylated
under the same conditions as those used for preparation
of 7 from 6, giving a residue that was purified by flash
chromatography (2.5:1 petroleum ether–EtOAc) to
D
anosyl-(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a-
D
anosyl-(1 ! 2)]-3,4-di-O-benzoyl-a-
D
(28)
furnish 26 (219 mg, 85.5%) as a foamy solid: ½aŠ þ46.6
D
1
(c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d 8.08–
7.26 (m, 45H, 9Ph), 6.10–5.81 (m, 9H, 4H-3, 4H-4,
Donor 11 (184 mg, 0.25 mmol) was coupled with
acceptor 27 (143 mg, 0.08 mmol) as described in the
general procedure, and the product was purified by
chromatography with 3:2 petroleum ether–EtOAc as the
000 ;2000
–CH2–CH@CH2), 5.65 (dd, 1H, J1
¼ 1:4 Hz,
000 ;3000
00 ;200
J2
¼ 3:2 Hz, H-2000), 5.59 (dd, 1H, J1
¼ 1:4 Hz,
¼ 3:2 Hz, H-200), 5.57 (dd, 1H, J1 ;2 ¼ 1:6 Hz,
eluent to give 28 (205 mg, 85.9%) as a foamy solid: ½aŠ
00 ;300
0
0
J2
0
D
J2 ;3 ¼ 3:2 Hz, H-20), 5.43–5.26 (m, 2H, –CH2–
À18.7 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
1
0
CH@CH2), 5.40 (dd, 1H, J1;2 ¼ 1:6 Hz, J2;3 ¼ 3:2 Hz, H-
8.16–6.81 (m, 85H, 17Ph), 6.28–5.90 (m, 15H, 2H-2, 6H-
3, 6H-4, –CH2–CH@CH2), 5.77 (s, 1H, H-2000), 5.70 (s,
1H, H-20), 5.48–5.29 (m, 2H, –CH2–CH@CH2), 5.34 (s,
2H, H-1000, H-100), 5.12 (s, 1H, H-10), 4.99 (s, 1H, H-1),
4.81–3.54 (m, 22H, 2H-2, 6H-5, 12H-6, –CH2–
CH@CH2), 2.10 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO);
2), 5.09 (d, 2H, J1
¼ 1:4 Hz, H-1000, H-100),
000 ;2000
00 ;200
¼ J1
4.93 (d, 1H, J1 ;2 ¼ 1:6 Hz, H-10), 4.87 (d, 1H,
J1;2 ¼ 1:6 Hz, H-1), 4.41–3.48 (m, 18H, 4H-5, 8H-6,
2CH2ClCO, –CH2–CH@CH2), 2.18 (s, 3H, CH3CO),
2.09 (s, 3H, CH3CO); 13C NMR (100 MHz, CDCl3): d
170.13, 169.68 (CH3CO), 166.69, 166.79 (CH2ClCO),
165.94, 165.50, 165.49, 165.46, 165.34, 165.32, 165.30,
165.25, 165.23 (PhCO), 97.89, 97.86, 97.16, 96.29 (C-1),
71.62, 71.29, 70.17, 70.12, 70.03, 69.89, 69.83, 69.71,
69.66, 68.88, 68.82, 66.61, 66.56, 66.20, 66.13, 66.06,
65.93, 65.69, 62.87, 40.80, 20.72, 20.68. Anal. Calcd for
C98H88Cl2O34: C 68.59; H 4.72. Found: C 68.88; H 4.66.
0
0
13C NMR (100 MHz, CDCl3):
d 169.99, 169.50
(CH3CO), 166.17, 165.99, 165.81, 165.79, 165.73,
165.54, 165.51, 165.19, 164.90, 164.84, 164.80, 164.77,
164.74, 164.68 (PhCO), 99.93, 99.75, 98.32, 97.96, 97.89,
97.68 (C-1), 71.46, 70.81, 70.70, 70.43, 70.33, 70.19,
70.07, 69.96, 69.82, 69.74, 69.33, 68.72, 68.64, 67.10,
66.99, 66.76, 66.58, 66.46, 66.10, 65.76, 62.93, 62.66,
20.69, 20.65. Anal. Calcd for C162H138O50: C 67.45; H
4.82. Found: C 67.63; H 4.78.
3.21. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(1 ! 6)-3,4-di-O-benzoyl-a- -mannopyranos-
-mannopyr-
-mannopyranoside
D-manno-
D
yl-(1 ! 6)-2-O-acetyl-3,4-di-O-benzoyl-a-
D
3.23. Allyl 3,4,6-tri-O-benzoyl-a-
(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a-
(1 ! 2)]-3,4-di-O-benzoyl-a- -mannopyranosyl-(1 ! 6)-
3,4-di-O-benzoyl-a-
-mannopyranosyl-(1 ! 6)-[2,3,4,6-
tetra-O-benzoyl-a-
-mannopyranosyl-(1 ! 2)]-3,4-di-O-
benzoyl-a- -mannopyranoside (29)
D
-mannopyranosyl-
anosyl-(1 ! 6)-3,4-di-O-benzoyl-a-
D
D-mannopyranosyl-
(27)
D
D
Dechloroacetylation of 26 (208 mg, 0.1 mmol) using the
same conditions as those used for preparation of 8 from
7 gave a residue that was purified by flash chromato-
graphy (2:1 petroleum ether–EtOAc) to furnish 27
D
D
Deacetylation of compound 28 (193 mg, 0.07 mmol) was
carried out under the same conditions as those used for
preparation of 13 from 12, giving a crude product that
(157 mg, 82.3%) as a foamy solid: ½aŠ þ37.5 (c 1.0,
D
1
CHCl3); H NMR (400 MHz, CDCl3): d 8.10–7.23 (m,

1770
Z. Ma et al. / Carbohydrate Research 339 (2004) 1761–1771
was purified by flash chromatography (2:1 petroleum
ether–EtOAc) to give 29 (144 mg, 76.8%) as a foamy
70.04, 69.84, 69.70, 69.51, 69.46, 69.37, 69.25, 68.61,
68.31, 67.80, 67.34, 67.21, 66.92, 66.63, 66.48, 66.37,
65.35, 64.27, 63.50, 63.31, 63.14, 63.01. Anal. Calcd for
C264H218O78: C 68.36; H 4.74. Found: C 68.69; H 4.73.
1
solid: ½aŠ þ47.3 (c 1.0, CHCl3); H NMR (400 MHz,
D
CDCl3): d 8.07–6.90 (m, 85H, 17Ph), 6.37 (dd, 1H,
J3;4 ¼ J4;5 ¼ 9:9 Hz, H-4), 6.15–5.76 (m, 14H, 2H-2, 6H-
3, 5H-4, –CH2–CH@CH2), 5.48 (s, 1H, H-1), 5.36 (s,
1H, H-1), 5.34–5.15 (m, 2H, –CH2–CH@CH2), 5.30 (s,
1H, H-1), 5.24 (s, 1H, H-1), 5.15 (s, 1H, H-1), 5.04 (s,
1H, H-1), 4.88–3.58 (m, 24H, 4H-2, 6H-5, 12H-6,
–CH2–CH@CH2); 13C NMR (100 MHz, CDCl3): d
166.17, 166.12, 166.03, 165.95, 165.73, 165.61, 165.51,
165.48, 165.40, 165.36, 165.08, 165.00, 164.78, 164.69,
164.60 (PhCO), 100.28, 100.05, 99.29, 97.85, 97.82,
97.54 (C-1), 73.23, 72.72, 71.43, 70.59, 70.26, 69.93,
69.83, 69.72, 69.63, 69.54, 69.50, 69.20, 68.94, 68.57,
68.38, 67.17, 67.11, 66.85, 66.79, 66.67, 66.56, 66.52,
66.25, 66.90, 65.56, 63.66, 62.94, 62.86, 62.69. Anal.
Calcd for C158H134O48: C 67.76; H 4.82. Found: C 67.48;
H 4.91.
3.25. Allyl [a-
D
-arabinofuranosyl-(1 ! 2)-a-
D-mannopyr-
D-
anosyl-(1 ! 2)]-a-
D
-mannopyranosyl-(1 ! 6)-[a-
mannopyranosyl-(1 ! 2)]-a- -mannopyranosyl-(1 ! 6)-
[a-
D
-arabinofuranosyl-(1 !D2)-a-
D
-mannopyranosyl-
-mannopyr-
(1 ! 2)]-a-
D
-mannopyranosyl-(1 ! 6)-[a-
D
anosyl-(1 ! 2)]-a-
D-mannopyranoside (31)
Decasaccharide 30 (133 mg, 0.03 mmol) was dissolved in
satd NH3–MeOH (30 mL). After 96 h at rt, the reaction
mixture was concentrated, and the residue was purified
by chromatography on Sephadex LH-20 (MeOH) to
afford 31 (43 mg, 92.5%) as a foamy solid: ½aŠ þ64.6 (c
D
1
1.0, H2O); H NMR (400 MHz, D2O): d 5.91–5.81 (m,
1H, –CH2–CH@CH2), 5.25 (d, 1H, J ¼ 17:2 Hz, –CH2–
CH@CHtrans), 5.18 (d, 1H, J ¼ 10:0 Hz, –CH2–
CH@CHcis), 5.09 (s, 1H, Manp H-1), 5.08 (s, 1H, Manp
H-1), 5.07 (s, 2H, Araf 2H-1), 5.02 (s, 1H, Manp H-1),
5.01 (s, 1H, Manp H-1), 4.99 (s, 2H, Manp 2H-1), 4.93
(s, 1H, Manp H-1), 4.91 (s, 1H, Manp H-1); 13C NMR
(100 MHz, D2O): d 109.36 (2C, Araf 2C-1), 102.32,
102.19, 101.32, 101.30, 98.17, 98.03, 97.53 (8C, Manp
8C-1), 83.65, 83.62, 81.20, 78.85, 78.70, 78.55, 77.46,
77.43, 76.46, 73.33, 73.26, 73.18, 72.85, 71.28, 71.19,
71.01, 70.64, 70.57, 70.49, 70.44, 70.24, 70.08, 70.04,
68.44, 67.02, 66.81, 66.75, 66.67, 66.48, 65.75, 65.63,
61.18, 61.15, 61.10, 61.07, 60.94, 60.86. Anal. Calcd for
C61H102O49: C 45.24; H 6.35. Found: C 45.13; H 6.33.
3.24. Allyl [2,3,5-tri-O-benzoyl-a-
(1 ! 2)-3,4,6-tri-O-benzoyl-a- -mannopyranosyl-
(1 ! 2)]-3,4,6-tri-O-benzoyl-a- -mannopyranosyl-
(1 ! 6)-[2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl-
(1 ! 2)]-3,4-di-O-benzoyl-a- -mannopyranosyl-(1!6)-
[2,3,5-tri-O-benzoyl-a-
-arabinofuranosyl-(1 ! 2)-3,4,6-
tri-O-benzoyl-a-
D-arabinofuranosyl-
D
D
D
D
D
D
-mannopyranosyl-(1 ! 2)]-3,4-di-O-
benzoyl-a-
benzoyl-a-
D
-mannopyranosyl-(1 ! 6)-[2,3,4,6-tetra-O-
D
-mannopyranosyl-(1 ! 2)]-3,4-di-O-benzoyl-
a-D-mannopyranoside (30)
Donor 18 (130 mg, 0.12 mmol) was coupled with
acceptor 29 (130 mg, 0.05 mmol) as described in the
general procedure, and the product was purified by
chromatography with 3:2 petroleum ether–EtOAc as the
Acknowledgements
eluent to give 30 (143 mg, 66.7%) as a foamy solid: ½aŠ
D
1
À13.7 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
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).
8.08–6.50 (m, 145H, 29Ph), 6.38–5.86 (m, 19H, Manp
2H-2, 8H-3, 8H-4, –CH2–CH@CH2), 5.61 (s, 1H, Araf
H-2), 5.58 (s, 1H, Araf H-2), 5.57 (s, 1H, Manp H-1),
5.52 (d, 1H, J3;4 ¼ 4:4 Hz, Araf H-3), 5.50 (d, 1H,
J3;4 ¼ 5:0 Hz, Araf H-3), 5.49 (s, 2H, Araf 2H-1), 5.46–
5.27 (m, 2H, –CH2–CH@CH2), 5.44 (s, 1H, Manp H-1),
5.34 (s, 1H, Manp H-1), 5.33 (s, 1H, Manp H-1), 5.28 (s,
1H, Manp H-1), 5.27 (s, 1H, Manp H-1), 5.22 (s, 1H,
Manp H-1), 5.11 (s, 1H, Manp H-1), 5.05 (s, 1H, Manp
H-2), 4.97 (s, 1H, Manp H-2), 4.89 (s, 1H, Manp H-2),
4.82–3.47 (m, 35H, Manp 3H-2, 8H-5, 16H-6, Araf
2H-4, 4H-5, –CH2–CH@CH2); 13C NMR (100 MHz,
CDCl3): d 166.25, 166.13, 166.09, 166.06, 165.95, 165.88,
165.82, 165.67, 165.65, 165.60, 165.55, 165.50, 165.45,
165.21, 165.14, 165.07, 165.02, 164.90, 164.81, 164.45,
164.39 (PhCO), 107.17, 106.71 (Araf C-1), 100.72,
100.28, 99.99, 99.43, 99.25, 98.05, 98.65, 98.02 (C-1),
82.08, 81.98, 81.75, 81.62, 76.32, 75.96, 75.25, 74.07,
72.58, 72.36, 71.86, 71.35, 71.07, 70.56, 70.45, 70.25,
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