A highly efficient synthesis of an octasaccharide, the repeating unit of the cell-wall mannan of Trichophyton mentagrophytes and T. rubrum

Article abstract of DOI:10.1016/S0008-6215(02)00117-9

A highly concise and effective synthesis of the mannose octasaccharide repeating unit of the cell-wall mannan of Trichophyton mentagrophytes and T. rubrum was achieved via 6-O-glycosylation of a tetrasaccharide acceptor with a tetrasaccharide donor, follo

Full text of DOI:10.1016/S0008-6215(02)00117-9

Carbohydrate Research 337 (2002) 1159–1164
www.elsevier.com/locate/carres
A highly efficient synthesis of an octasaccharide, the repeating unit
of the cell-wall mannan of Trichophyton mentagrophytes and T.
rubrum
Jun Ning,a Linsen Heng,b Fanzuo Konga,*
aResearch Center for Eco-En6ironmental Sciences, Chinese Academy of Sciences, PO Box 2871, Beijing 100085, PR China
bDaxian Normal College, Department of Chemistry, Sichuan, PR China
Received 25 March 2002; accepted 30 April 2002
Abstract
A highly concise and effective synthesis of the mannose octasaccharide repeating unit of the cell-wall mannan of Trichophyton
mentagrophytes and T. rubrum was achieved via 6-O-glycosylation of a tetrasaccharide acceptor with a tetrasaccharide donor,
followed by deprotection. The key tetrasaccharide (11) was constructed by selective 6-O-glycosylation of allyl 3,4-di-O-benzoyl-a-
D
-mannopyranosyl-(1“6)-2,3,4-tri-O-benzoyl-a-
D
-mannopyranoside with 6-O-acetyl-2,3,4-tri-O-benzoyl-a-
D-mannopyranosyl
-mannopyranosyl trichloroacetimidate. The tetrasaccharide acceptor
trichloroacetimidate, then with 2,3,4,6-tetra-O-benzoyl-a-
(13) was obtained by selective 6-O-deacetylation of 11, while the tetrasaccharide donor 12 was obtained by deallylation of 11,
D
followed by trichloroacetimidation. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Regio- and stereoselective synthesis; Mannopyranosyl oligosaccharides; Trichloroacetimidates
1. Introduction
The anthropophilic dermatophytes, Trichophyton
mentagrophytes and T. rubrum, cause chronic, relatively
uninflamed, skin infections of the feet (tinea pedis) and
Blake et al. examined the ability of a cell-wall man-
nan isolated from T. rubrum to suppress cell-mediated
immune reactions4 and found that the T. rubrum man-
nan inhibits the lymphoproliferative response of human
mononuclear leukocytes to a variety of antigenic and
mitogenic stimuli in vitro. Furthermore, Grando et al.
demonstrated that the target cells for this effect of the
mannan are monocytes and keratinocytes, by monitor-
ing the binding and uptake of FITC-mannan using flow
cytometry and fluorescence microscopy.5,6 To further
elucidate the molecular structure responsible for the
immunoinhibitory activity of the fungi mannan, it will
be very useful to synthesize the repeating unit of the
mannan. We report herewith a highly efficient and
concise synthesis of the octasaccharide 1, the repeating
unit of the cell-wall mannan of T. mentagrophytes and
T. rubrum.
body (tinea corporis), often associated with infection of
the nails (onychoycosis). About 90% of chronic derma-
tophyte infections are caused by T. mentagrophytes and
T. rubrum,1 in part because these organisms can sup-
press inflammation and cell-mediated immunity.2 The
cell-wall polysaccharides of these fungi are known to be
the major immunologically active substances. There are
mainly two kinds of polysaccharides present in the
cell-wall, i.e., mannan and galactomannan.3 The man-
nan has a linear backbone consisting of a-(1“6)-linked
mannose units, with a-(1“2)-linked mannose units as
side chains, and its structure3 is shown as follows:
* Corresponding author. Tel.: +86-10-62936613; fax: +
86-10-62923563
E-mail address: fzkong@mail.rcees.ac.cn (F. Kong).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00117-9

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J. Ning et al. / Carbohydrate Research 337 (2002) 1159–1164
structure of the tetrasaccharide 11. Selective removal of
the 6-O-acetyl group of the tetrasaccharide 11 gave the
glycosyl acceptor 13. Deallylation9 of 11 with PdCl2,
followed by activation with CCl3CN in the presence of
K2CO3, gave the tetrasaccharide donor 12. The fully
protected octasaccharide 14 was smoothly obtained by
coupling 13 with 12. The 13C NMR spectrum of 14
gave eight signals for C-1 (100.08, 100.06, 98.9, 98.7,
98.1, 97.8, 97.6, 96.8 (8 C-1). Finally, deacylation of 14
in ammonia–saturated methanol gave the target oc-
tasaccharide 1. A bioassay of 1 for its immunoin-
hibitory activity is in progress.
In summary, a highly efficient and concise synthesis
of the mannose octasaccharide repeating unit of the
mannans of the cell-walls of some fungi was achieved
by regio- and stereoselective glycosylation using glyco-
syl trichloroacetimidates as the donors and partially
protected sugars as the acceptors. The sole use of acyl
groups in the synthesis substantially simplified the pro-
cedure. This method should also be useful for the
synthesis of high-mannose oligosaccharides.
2. Results and discussion
First we synthesized the key synthon 6 (Scheme 1).
Thus, tritylation of 1,2-O-(R-ethylidene)-b-D-manno-
pyranose7 (2), followed by benzoylation in a one-pot
manner, gave the 3,4-di-O-benzoyl-1,2-O-(R-ethyli-
dene)-6-O-trityl-
with CH2Cl2–AcOH–Ac2O–H2SO4 in
1:1:0.6:0.18 afforded the 1,2,6-tri-O-acetyl-3,4-di-O-
-mannopyranose (4). The triacetate 4 was
D
-mannopyranose (3). Acetolysis of 3
a
ratio of
benzoyl-a-
D
selectively deacetylated at the anomeric position with
benzylamine in THF in high yield to give the corre-
sponding 2,6-di-O-acetyl-3,4-di-O-benzoyl-D-mannopy-
ranose (5). Subsequent reaction of 5 with CCl3CN–
DBU in dichloromethane afforded the glycosyl donor
6.
With the synthon 6 in hand, construction of the
target compound was readily carried out. As shown in
Scheme 2, the disaccharide 7 was prepared using 6 as
3. Experimental
the glycosyl donor and allyl 2,3,4-tri-O-benzoyl-a-D-
mannopyranoside as the acceptor.8 Selective removal of
the acetyl groups of 7 in methanol solution containing
0.5% HCl gave the diol glycosyl acceptor 8.8 Coupling
General methods.—Optical rotations were deter-
mined at 25 °C with a Perkin–Elmer model 241-Mc
automatic polarimeter. Melting points were determined
1
of 8 with 6-O-acetyl-2,3,4-tri-O-benzoyl-a-
D
-mannopy-
with a ‘Mel-Temp’ apparatus. H NMR and 13C NMR
ranosyl trichloroacetimidate8 as the glycosyl donor se-
lectively gave the (1“6)-linked trisaccharide 9.
Acetylation of 9 confirmed the 6-O-glycosylation as the
1H NMR spectrum of acetylated trisaccharide 10
showed a newly emerged doublet of doublets at l 5.67
ppm for H-2. Condensation of 9 with 2,3,4,6-tetra-O-
spectra were recorded with Bruker ARX 400 spectrom-
1
eters (400 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 recorded with a VG PLATFORM mass
spectrometer in the ESI mode. Thin-layer chromatogra-
phy (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 chromatog-
raphy 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 B60 °C under
reduced pressure.
benzoyl-a-D
-mannopyranosyl trichloroacetimidate8 us-
ing TMSOTf as catalyst afforded the tetrasaccharide
1
11. The H NMR spectrum of 11 showed one acetyl
signal (l 1.96), allyl signals (5.47–5.30), and four H-1
signals (5.33, 5.20, 5.05, 4.91) characteristic of the
3,4-Di-O-benzoyl-1,2-O-[(R)-ethylidene]-6-O-trityl-
i -
D
- mannopyranose (3).—A solution of 1,2 - O - [(R)-
ethylidene]-b-
D
-mannopyranose (2)7 (6.4 g, 31.1 mmol)
and trityl chloride (9.8 g, 35.0 mmol) in pyridine (100
mL) was stirred at 50 °C for 32 h, at the end of which
time TLC (1:1 petroleum ether–EtOAc) indicated that
the reaction was complete. The reaction mixture was
cooled to 0 °C, and then benzoyl chloride (7.9 mL, 68
mmol) was added dropwise within 30 min to keep the
reaction temperature under 40 °C. After 24 h, water
(300 mL) was added to the reaction mixture, and
stirring was continued for 30 min. The mixture was
extracted with CH2Cl2 (3×100 mL), and the combined
Scheme 1. Conditions and reagents: (a) i. trityl chloride (1.2
equiv), pyridine, 50 °C, 32 h; ii. PhCOCl (4.8 equiv), B40 °C,
24 h (71% for two steps); (b) 1:1:0.6:0.18 CH2Cl2–HOAc–
Ac2O–H2SO4, rt, 20 h (83%); (c) benzylamine (4.0 equiv),
THF, rt, 24 h (88%); (d) CCl3CN (3.3 equiv), DBU (0.3
equiv), rt, 5 h (86%).

J. Ning et al. / Carbohydrate Research 337 (2002) 1159–1164
1161
Scheme 2. Conditions and reagents: (a) allyl 2,3,4-tri-O-benzoyl-a-
rt, 3 h (88%); (b) MeOH–0.5% HCl, rt, 12–14 h (93–96%); (c) 6-O-acetyl-2,3,4-tri-O-benzoyl-a-
timidate (1.0 equiv), CH2Cl2, TMSOTf (0.08 equiv), rt, 3 h (86%); (d) (Ac)2O, pyridine, rt, 5 h (100%); (e) 2,3,4,6-tetra-O-benzoyl-
-mannopyranosyl trichloroacetimidate (1.4 equiv), CH2Cl2, TMSOTf (0.16 equiv), rt, 3 h (85%); (f) i. PdCl2, CH3OH–CH2Cl2,
D
-mannopyranoside (1 equiv), TMSOTf (0.26 equiv), CH2Cl2,
D
-mannopyranosyl trichloroace-
a-
D
2 h; ii: CCl3CN, DBU, CH2Cl2 8 h (78%); (g) 12 (1.2 equiv), CH2Cl2, TMSOTf (0.3 equiv), rt, 3 h (80%); (h) CH3OH satd with
dry NH3, rt, 72 h (98%).
extracts were washed with 1 N HCl and satd aq
NaHCO3, dried (Na2SO4) and concentrated to a syrup
that was subjected to column chromatography with 4:1
petroleum ether–EtOAc as the eluent. Compound 3
(14.5 g, 71%) was obtained as a syrup: [h]D −37.2° (c
ice bath, and H2SO4 (8.8 mL) was added dropwise over
20 min. After the addition was complete, the ice bath was
removed and the reaction was allowed to continue for 20
h at ambient temperature. The reaction solution was
poured into ice water (400 mL), stirring was continued
for an additional 15 min, and the mixture was extracted
with CHCl3 (3×100 mL). The combined extracts were
washed with 10% aq NaHCO3 (3×60 mL), dried over
Na2SO4, and concentrated to a syrup that was subjected
to column chromatography with 4:1 petroleum ether–
EtOAc as the eluent. Compound 4 (7.2 g, 83%) was
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l 8.01, 7.90
(2 d, 4 H, BzH), 7.65–7.08 (m, 21 H, PhH), 5.97 (dd,
1 H, J3,4=J5,4 10.0 Hz, H-4), 5.48 (dd, 1 H, J2,3 3.6 Hz,
J4,3 10.0 Hz, H-3), 5.43 (d, 1 H, J2,1 2.0 Hz, H-1), 5.37
(q, J 4.8 Hz, CH-CH3), 4.49 (dd, J1,2 2.0 Hz, J3,2 3.6 Hz,
H-2), 3.79 (m, 1 H, J6a,5 2.8 Hz, J6b,5 4.4 Hz, J4,5 10.0
Hz, H-5), 3.37 (dd, 1 H, J6a,6b 10.2 Hz, J5,6a 2.8 Hz,
H-6a), 3.22 (dd, 1 H, J6a,6b 10.2 Hz, J5,6b 4.4 Hz, H-6b),
1.60 (d, 3 H, J 4.8 Hz, CH-CH3). Anal. Calcd for
C41H36O8: C, 74.99; H, 5.52. Found: C, 75.16; H, 5.55.
1
obtained as a syrup: [h]D −28.2° (c 1.0, CHCI3); H
NMR (400 MHz, CDCl3): l 7.95, 7.89 (2 d, 4 H, BzH),
7.53–7.34 (m, 6 H, BzH), 6.21 (d, 1 H, J2,1 1.9 Hz, H-1),
5.84 (dd, 1 H, J3,4=J5,4 9.9 Hz, H-4), 5.75 (dd, 1 H, J2,3
4.7 Hz, J4,3 9.9 Hz, H-3), 5.48 (dd, 1 H, J1,2 1.9 Hz, J3,2
4.7 Hz, H-2), 4.36–4.20 (m, 3 H, H-5, H-6a, H-6b), 2.24,
2.18, 2.05 (3 s, 9 H, 3 COCH3). Anal. Calcd for
C26H26O11: C, 60.70; H, 5.09. Found: C, 60.98; H, 5.15.
1,2,6-Tri-O-acetyl-3,4-di-O-benzoyl-h-D-mannopyran-
ose (4).—Compound 3 (11 g, 16.77 mol) was dissolved
in a mixture of CH2Cl2 (50 mL), Ac2O (50 mL) and
AcOH (30 mL), the solution was cooled to 10 °C in an

1162
J. Ning et al. / Carbohydrate Research 337 (2002) 1159–1164
2,6-Di-O-acetyl-3,4-di-O-benzoyl-h-
D
-mannopyranose
containing 0.5% HCl was stirred at rt for 12 h, at the end
of which time TLC (2:1 petroleum ether–EtOAc) indi-
cated that the starting material had disappeared. The
mixture was neutralized with Et3N and then concen-
trated to dryness. The residue was partitioned between
water and CH2Cl2, then 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 8 as a syrup (1.36 g, 93%): [h]D −8.8° (c
(5).—A solution of compound 4 (5 g, 9.73 mmol) and
benzylamine (3 mL, 27.4 mmol) in anhyd THF (30 mL)
was stirred at rt for 24 h, at the end of which time TLC
(3:1 petroleum ether–EtOAc) indicated that the reaction
was complete. The solution was then concentrated.
Purification by flash column chromatography on silica
gel (3:1 petroleum ether–EtOAc) gave compound 5 as a
1
syrup (3.96 g, 88%): [h]D −37.4° (c 0.6, CHCl3); H
1
1.0, CHCl3); H NMR (400 MHz, CDCl3): l 8.15, 8.05,
NMR (400 MHz, CDCl3): l 7.95, 7.89 (2 d, 4 H, BzH),
7.52–7.32 (m, 6 H, BzH), 5.84–5.76 (m, 2 H, H-3, H-4),
5.50 (dd, 1 H, J1,2 1.8 Hz, J3,2 2.9 Hz, H-2), 5.38 (d, 1
H, J2,1 1.8 Hz, H-1), 4.51–4.24 (m, 3 H, H-5, H-6a, H-6),
2.17, 2.06 (2 s, 6 H, 2 COCH3). Anal. Calcd for
C24H24O10: C, 61.01; H, 5.12. Found: C, 61.29; H, 5.19.
8.00, 7.96, 7.89 (5 d, 10 H, BzH), 7.53–7.25 (m, 15 H,
BzH), 6.06–5.71 (m, 6 H, CHꢀCH2, 2 H-4, 2 H-3, 1
H-2), 5.48 (dd, 1 H, 2J 1.4 Hz, 3Jtrans 17.2 Hz, CHꢀCH2),
2
3
5.38 (dd, 1 H, J 1.4 Hz, Jcis 10.4 Hz, CHꢀCH2), 5.15
(d, 1 H, J2,1 1.5 Hz, H-1), 5.05 (d, 1 H, J2%,1% 1.4 Hz, H-1%),
4.41–3.46 (m, 9 H, CH2CHꢀCH2, 2 H-5, 4 H-6, 1 H-2).
Anal. Calcd for C50H46O16: C, 66.51; H, 5.13. Found: C,
66.80; H, 5.19.
2,6-Di-O-acetyl-3,4-di-O-benzoyl-h-D-mannopyranosyl
trichloroacetimidate (6).—A mixture of 5 (4.0 g, 8.47
mmol), CCl3CN (2.1 mL, 20.9 mmol), and 1,8-diazabicy-
clo[5.4.0]undecene (DBU) (0.25 mL, 1.67 mmol) in dry
CH2Cl2 (25 mL) was stirred under N2 for 5 h and then
concentrated. The residue was purified by flash chro-
matography (4:1 petroleum ether–EtOAc) to give 6 (4.5
Allyl 6-O-acetyl-2,3,4-tri-O-benzoyl-h-
ranosyl-(1“6)-3,4-di-O-benzoyl-h- -mannopyranosyl-
(1“6)-2,3,4-tri-O-benzoyl-h- -mannopyranoside (9).—
D-mannopy-
D
D
A solution of 8 (0.37 g, 0.41 mmol) and TMSOTf (10 mL,
0.055 mmol) in dry CH2Cl2 (6 mL) was stirred with dried
1
g, 86%) as a syrup: [h]D −19.8° (c 1.0, CHCl3); H
,
molecular sieves (4 A, 0.4 g) under N2 for 15 min, and
then 6-O-acetyl-2,3,4-tri-O-benzoyl-a- -mannopyran-
NMR (400 MHz, CDCl3): l 8.84 (s, 1 H, NH), 7.96, 7.89
(2 d, 4 H, BzH), 7.53–7.34 (m, 6 H, BzH), 6.41 (d, 1 H,
J2,1 1.8 Hz, H-1), 5.89 (dd, 1 H, J3,4=J5,4 10.0 Hz, H-4),
5.80 (dd, 1 H, J2,3 3.4 Hz, J4,3 10.0 Hz, H-3), 5.70 (dd,
1 H, J1,2 1.8 Hz, J3,2 3.4 Hz, H-2), 4.46–4.26 (m, 3 H,
H-5, H-6a, H-6b), 2.18, 2.04 (2 s, 6 H, 2 COCH3). Anal.
Calcd for C26H24Cl3NO10: C, 50.62; H, 3.92. Found: C,
50.79; H, 3.85.
D
osyl trichloroacetimidate (0.28 g, 0.41 mmol) in CH2Cl2
(4 mL) was added dropwise within 20 min. After 3 h the
reaction mixture was diluted with CH2Cl2 (30 mL) and
washed with satd aq NaHCO3 (5 mL). The organic layer
was dried over Na2SO4 and concentrated. Purification of
the residue by flash chromatography (2:1 petroleum
ether–EtOAc) gave 9 as a syrup (0.50 g, 86%): [h]D
Allyl 2,6-di-O-acetyl-3,4-di-O-benzoyl-h-
ranosyl-(1“6)-2,3,4-tri-O-benzoyl-h- -mannopyrano-
side (7).—A solution of 6 (2.5 g, 4.06 mmol) and allyl
2,3,4-tri-O-benzoyl-a- -mannopyranoside (2.16 g, 4.0
mmol) in dry CH2Cl2 (40 mL) was stirred with dried
D-mannopy-
1
−51.4° (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): l
D
8.08–7.25 (m, 40 H, 8 BzH), 6.26–5.77 (m, 9 H,
CHꢀCH2, 3 H-4, 3 H-3, 2 H-2), 5.41 (dd, 1 H, 2J 1.4 Hz,
D
2
3
3Jtrans 17.2 Hz, CHꢀCH2), 5.25 (dd, 1 H, J 1.4 Hz, Jcis
10.4 Hz, CHꢀCH2), 5.20 (d, 1 H, J2,1 1.4 Hz, H-1), 5.14
(d, 1 H, J2,1 1.3 Hz, H-1), 4.84 (d, 1 H, J 1.4 Hz, H-1),
4.47–3.37 (m, 13 H, CH2CHꢀCH2, 1 H-2, 3 H-5, 6 H-6,
OH), 2.00 (s, 3 H, COCH3). Anal. Calcd for C79H70O25:
C, 66.85; H, 4.97. Found: C, 66.98; H, 4.86.
,
molecular sieves (4 A, 1 g) under N2 for 15 min, and then
TMSOTf (0.2 mL, 1.1 mmol) was added dropwise. After
1 h the reaction mixture was diluted with CH2Cl2 (50
mL) and washed with satd aq NaHCO3 (15 mL). The
organic layer was dried over Na2SO4 and concentrated.
Purification of the residue by flash chromatography (3:1
petroleum ether–EtOAc) gave 7 as a syrup (3.47 g, 88%):
[h]D −51.4° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): l 8.14, 7.99, 7.97, 7.89, 7.84 (5 d, 10 H, BzH),
7.55–7.27 (m, 15 H, BzH), 5.99 (m, 1 H, CHꢀCH2),
5.97–5.73 (m, 5 H, 2 H-4, 2 H-3, H-2), 5.54 (dd, 1 H,
H-2), 5.53 (dd, 1 H, 2J 1.3 Hz, 3Jtrans 17.1 Hz, CHꢀCH2),
Allyl 6-O-acetyl-2,3,4-tri-O-benzoyl-h-
ranosyl-(1“6)-2-O-acetyl-3,4-di-O-benzoyl-h-
nopyranosyl-(1“6)-2,3,4-tri-O-benzoyl-h- -mannopy-
D
-mannopy-
D
-man-
D
ranoside (10).—To a solution of 9 (1.20 g, 0.84 mmol)
in pyridine (20 mL), Ac2O (1 mL, 10 mmol) was added
dropwise, and the mixture was stirred overnight at rt.
TLC (2:1 petroleum ether–EtOAc) indicated that the
reaction was complete. The mixture was diluted with
CH2Cl2 and sequentially washed with 1 N HCl, water,
and satd aq NaHCO3. The organic layers were com-
bined, dried, and concentrated. Purification by column
chromatography (2:1 petroleum ether–EtOAc) quanti-
tatively gave 10 as a syrup: [h]D −99.6° (c 1.0, CHCl3);
1H NMR (400 MHz, CDCl3): l 8.08–7.24 (m, 40 H, 8
BzH), 6.21–5.79 (m, 9 H, CHꢀCH2, 3 H-4, 3 H-3, 2
2
3
5.35 (dd, 1 H, J 1.3 Hz, Jcis 10.4 Hz, CHꢀCH2), 5.16
(d, 1 H, J2%,1% 1.5 Hz, H-1%), 4.97 (d, 1 H, J2,1 1.5 Hz, H-1),
4.40–3.70 (m, 8 H, CH2CHꢀCH2, 2 H-5, 4 H-6), 2.13,
1.88 (2 s, 6 H, 2 COCH3). Anal. Calcd for C54H50O18:
C, 65.72; H, 5.10. Found: C, 65.94; H, 5.07.
Allyl 3,4-di-O-benzoyl-h-
2,3,4-tri-O-benzoyl-h- -mannopyranoside (8).—A solu-
tion of 7 (1.6 g, 1.62 mmol) in aq MeOH (80 mL)
D
-mannopyranosyl-(1“6)-
D
2
H-2), 5.67 (dd, 1 H, H-2), 5.41 (dd, 1 H, J 1.4 Hz,

J. Ning et al. / Carbohydrate Research 337 (2002) 1159–1164
1163
2
3
3Jtrans 17.2 Hz, CHꢀCH2), 5.25 (dd, 1 H, J 1.4 Hz, Jcis
10.4 Hz, CHꢀCH2), 5.20 (d, 1 H, J2,1 1.4 Hz, H-1), 5.06
(d, 1 H, J2,1 1.3 Hz, H-1), 4.87 (d, 1 H, J2,1 1.4 Hz,
H-1), 4.42–3.42 (m, 11 H, CH2CHꢀCH2, 3 H-5, 6 H-6),
2.21, 2.00 (2 s, 6 H, 2 COCH3). Anal. Calcd for
C81H72O26: C, 66.57; H, 4.96. Found: C, 66.31; H, 5.03.
Hz, H-1), 5.05 (d, 1 H, J 1.4 Hz, H-1), 4.90 (d, 1 H, J2,1
1.2 Hz, H-1), 4.71–3.48 (m, 15 H, CH2CHꢀCH2, H-2, 4
H-5, 8 H-6); 13C NMR (100 MHz, CDCl3): l 166.6,
166.0, 165.7, 165.6, 165.5, 165.5, 165.4, 165.4, 165.2,
164.9, 164.9, 164.7 (12 C, 12 COPh), 133.4–127.8 (73
C, 12 Ph, CH2CHꢀCH2), 118.2 (1 C, CH2CHꢀCH2),
99.9, 98.7, 97.3, 96.8 (4 C-1). Anal. Calcd for
C111H94O33: C, 68.17; H, 4.84. Found: C, 68.44; H,
4.78.
Allyl 6-O-acetyl-2,3,4-tri-O-benzoyl-h-
ranosyl-(1“6)-[2,3,4,6-tetra-O-benzoyl-h-
ranosyl-(1“2)]-3,4-di-O-benzoyl-h-
(1“6)-2,3,4-tri-O-benzoyl-h- -mannopyranoside (11).
D
-mannopy-
-mannopy-
D
D
-mannopyranosyl-
D
6-O-Acetyl-2,3,4-tri-O-benzoyl-h-
D
-mannopyranosyl-
-mannopyranosyl-
—A solution of 9 (0.37 g, 0.26 mmol) and TMSOTf (10
mL, 0.055 mmol) in dry CH2Cl2 (6 mL) was stirred with
dried molecular sieves (4 A, 0.4 g) under N2 for 15 min,
and then 2,3,4,6-tetra-O-benzoyl-a- -mannopyranosyl
(1“6)-[2,3,4,6-tetra-O-benzoyl-h-
D
(1“2)]-3,4-di-O-benzoyl-h-
D
-mannopyranosyl-(1“6)-
,
2,3,4-tri-O-benzoyl-h- -mannopyranosyl trichloroacet-
D
D
imidate (12).—A mixture of compound 11 (2.24 g, 1.12
mmol) and PdCl2 (50 mg) in dry MeOH (50 mL)
(Caution! Extreme fire hazard.) was stirred vigorously
for 4 h at rt, TLC (1:1 petroleum ether–EtOAc) indi-
cated 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 (20 mL), then CCl3CN (0.3 mL, 3
mmol) and DBU (42 mL, 0.3 mmol) were added. The
reaction mixture was stirred for 2 h, at the end of which
time TLC (1:1 petroleum ether–EtOAc) indicated that
the reaction was complete. Concentration of the reac-
tion mixture, followed by purification on a silica gel
column with 1:1 petroleum ether–EtOAc as the eluent,
furnished the tetrasaccharide donor 12 in good yield
(1.84 g, 78% two steps): [h]D −71.5° (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): l 8.99 (s, 1 H, NH), 8.09–
7.03 (m, 60 H, 12 BzH), 6.64 (d, 1 H, J2,1 1.5 Hz, H-1),
6.35–5.90 (m, 11 H, 4 H-4, 4 H-3, 3 H-2), 5.54 (d, 1 H,
J2,1 1.2 Hz, H-1), 5.31 (d, 1 H, J2,1 1.3 Hz, H-1), 5.12
(d, 1 H, J2,1 1.4 Hz, H-1), 4.88 (d, 1 H, J2,1 1.5 Hz,
H-1), 4.78–3.47 (m, 13 H, H-2, 4 H-5, 8 H-6), 2.04 (s,
3 H, COCH3); 13C NMR (100 MHz, CDCl3): l 170.4 (1
C, COCH3), 165.9, 165.6, 165.6, 165.5, 165.4, 165.3,
165.3, 165.2, 165.0, 164.9, 164.8, 164.7 (12 C, 12
COPh), 159.7 (1 C, CCl3), 133.5–127.7 (72 C, 12 Ph),
99.9, 98.7, 97.1, 94.6 (4 C-1), 90.1 (1 C, C(NH)CCl3),
20.4 (1 C, COCH3). Anal. Calcd for C112H92Cl3NO34:
C, 63.99; H, 4.41. Found: C, 63.71; H, 4.48.
trichloroacetimidate (0.26 g, 0.35 mmol) in CH2Cl2 (4
mL) was added dropwise within 20 min. After 3 h the
reaction mixture was diluted with CH2Cl2 (30 mL) and
washed with satd aq NaHCO3 (5 mL). The organic
layer was dried over Na2SO4 and concentrated in
vacuo. Purification of the residue by flash chromatogra-
phy (2:1 petroleum ether–EtOAc) gave 11 as a syrup
1
(0.45 g, 85%): [h]D −72.5° (c 1.0, CHCl3); H NMR
(400 MHz, CDCl3): l 8.10–7.02 (m, 60 H, 12 BzH),
6.20–5.67 (m, 12 H, CHꢀCH2, 4 H-4, 4 H-3, 3 H-2),
2
3
5.47 (dd, 1 H, J 1.5 Hz, Jtrans 17.1 Hz, CHꢀCH2), 5.33
2
3
(d, 1 H, J2,1 1.2 Hz, H-1), 5.30 (dd, 1 H, J 1.5 Hz, Jcis
10.4 Hz, CHꢀCH2), 5.20 (d, 1 H, J 1.4 Hz, H-1), 5.05
(d, 1 H, J2,1 1.4 Hz, H-1), 4.91 (d, 1 H, J2,1 1.4 Hz,
H-1), 4.77–3.49 (m, 15 H, CH2CHꢀCH2, H-2, 4 H-5, 8
H-6), 1.96 (s, 3 H, COCH3); 13C NMR (100 MHz,
CDCl3): l 170.5 (1 C, COCH3), 166.0, 165.7, 165.7,
165.6, 165.5, 165.5, 165.4, 165.3, 165.1, 164.9, 164.8,
164.7 (12 C, 12 COPh), 133.3–127.7 (73 C, 12 Ph,
CH2CHꢀCH2), 118.2 (1 C, CH2CHꢀCH2), 100.0, 98.8,
97.3, 96.8 (4 C-1), 20.5 (1 C, COCH3). Anal. Calcd for
C
113H96O34: C, 67.93; H, 4.84. Found: C, 67.78; H,
4.89.
Allyl 2,3,4-tri-O-benzoyl-h-
6)-[2,3,4,6-tetra-O-benzoyl-h -
2)]-3,4-di-O-benzoyl-h- -mannopyranosyl-(1“6)-2,3,4-
tri-O-benzoyl-h- -mannopyranoside (13).—A solution
D
-mannopyranosyl-(1“
D
-mannopyranosyl-(1“
D
D
of 11 (1.6 g, 0.80 mmol) in MeOH (80 mL) containing
0.5% HCl was stirred at rt for 12 h, at the end of which
time TLC (1:1 petroleum ether–EtOAc) indicated that
the starting material had disappeared. The mixture was
neutralized with Et3N and then concentrated to dry-
ness. The residue was partitioned between water and
CH2Cl2, then the organic layer was dried over Na2SO4,
and concentrated to a syrup. Purification of the residue
by flash chromatography (1:1 petroleum ether–EtOAc)
Allyl 6-O-acetyl-2,3,4-tri-O-benzoyl-h-
ranosyl-(1“6)-[2,3,4,6-tetra-O-benzoyl-h-
ranosyl-(1“2)]-3,4-di-O-benzoyl-h-
(1“6)-2,3,4-tri-O-benzoyl-h- -mannopyranosyl-(1“6)-
2,3,4 - tri - O - benzoyl - h - - mannopyranosyl - (1“6)-
[2,3,4,6-tetra-O-benzoyl-h- -mannopyranosyl-(1“2)]-
3,4-di-O-benzoyl-h- -mannopyranosyl-(1“6)-2,3,4-
tri-O-benzoyl-h- -mannopyranoside (14).—The tetra-
D
-mannopy-
D
-mannopy-
D
-mannopyranosyl-
D
D
D
D
D
1
gave 13 (1.50 g, 96%): [h]D −79.6° (c 1.0, CHCl3); H
saccharide donor 12 (1.19 g, 0.57 mmol) and the tetra-
saccharide acceptor 13 (1.07 g, 0.55 mmol) were dried
together under high vacuum for 2 h, then dissolved in
anhyd CH2Cl2 (30 mL). TMSOTf (30 mL) was added
dropwise at −20 °C with N2 protection. The reaction
mixture was stirred for 3 h, during which time the
NMR (400 MHz, CDCl3): l 8.11–7.08 (m, 60 H, 12
BzH), 6.21–5.60 (m, 12 H, CHꢀCH2, 4 H-4, 4 H-3, 3
3
H-2), 5.47 (dd, 1 H, 2J 1.4 Hz, Jtrans 17.2 Hz,
CHꢀCH2), 5.32 (d, 1 H, J2,1 1.2 Hz, H-1), 5.30 (dd, 1 H,
3
2J 1.4 Hz, Jcis 10.4 Hz, CHꢀCH2), 5.23 (d, 1 H, J2,1 1.3

1164
J. Ning et al. / Carbohydrate Research 337 (2002) 1159–1164
product 1 as a white amorphous powder (274 mg, 98%):
[h]D +47.1° (c 1.0, H2O); H NMR (D2O, 400 MHz):
reaction temperature gradually raised to ambient tem-
perature. Then the mixture was neutralized with Et3N
and concentrated under reduced pressure to an oily
residue. Purification by column chromatography (1:1
petroleum ether–EtOAc) gave 14 (1.72 g, 80%) as a
syrup: [h]D −66.2° (c 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): l 8.11–6.88 (m, 120 H, 24 BzH), 6.24–
5.76 (m, 23 H, CHꢀCH2, 8 H-4, 8 H-3, 6 H-2), 5.46 (dd,
1
l 5.87 (m, 1 H, CHꢀCH2), 5.24–5.14 (2 H, CHꢀCH2),
5.00–4.79 (8 H-1); 13C NMR (100 MHz, D2O): 133.3 (1
C, CH2CHꢀCH2), 118.2 (1 C, CH2CHꢀCH2), 100.2,
100.1, 99.2, 99.17, 99.10, 99.0, 97.8, 96.8 (8 C-1). ES-
IMS: Calcd for C51H86O41, 1355.22 [M]. Found, 1378.1
(M+Na)+.
2
3
1 H, J 1.5 Hz, Jtrans 17.1 Hz, CHꢀCH2), 5.41 (d, 1 H,
2
3
J2,1 1.2 Hz, H-1), 5.28 (dd, 1 H, J 1.5 Hz, Jcis 10.4 Hz,
CHꢀCH2), 5.25 (d, 1 H, J2,1 1.3 Hz, H-1), 5.17 (d, 1 H,
J2,1 1.3 Hz, H-1), 5.09 (d, 1 H, J2,1 1.2 Hz, H-1), 5.02
(d, 1 H, J2,1 1.2 Hz, H-1), 5.00 (m, 2 H, 2 H-1), 4.95 (d,
Acknowledgements
This work was supported by the Beijing Natural
Science Foundation (6021004) and National Natural
Science Foundation of China (59973026 and 29905004).
1
H, J2,1 1.2 Hz, H-1), 4.79–3.38 (m, 28 H,
CH2CHꢀCH2, 2 H-2, 8 H-5, 16 H-6), 2.03 (s, 3 H,
COCH3); 13C NMR (100 MHz, CDCl3): l 170.3 (1 C,
COCH3), 166.0–164.6 (24 C, 24 COPh), 133.3–127.3
(145 C, 24 Ph, CH2CHꢀCH2), 118.1 (1 C, CH2CHꢀ
CH2), 100.08, 100.06, 98.9, 98.7, 98.1, 97.8, 97.6, 96.8 (8
C-1), 20.3 (1 C, COCH3). Anal. Calcd for C221H184O66:
C, 68.13; H, 4.76. Found: C, 68.41; H, 4.70.
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