Synthesis of a hexasaccharide, the repeating unit of O-deacetylated GXM of C. neoformans serotype A

Article abstract of DOI:10.1016/S0008-6215(03)00264-7

β-D-GlcpA-(1→2)-α-D-Manp-(1→3)-[β-D-Xylp- (1→2)]-α-D-Manp-(1→3)[-β-D-Xylp-(1→2)]-α-D- Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A, was synthesized as its allyl glycoside. Thus, 3-O-selective acetylation of allyl 4,6-O-benzylidene-α-D-mannopyranoside afforded 2, and subsequent glycosylation of 2 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate furnished the β-(1→2)-linked disaccharide 4. Debenzylidenation followed by benzoylation gave allyl 2,3,4-tri-O-benzoyl-β-D-xylopyranosyl-(1→2)-3-O-acetyl-4,6-di-O- benzoyl-α-D-mannopyranoside (5), and selective 3-O-deacetylation gave the disaccharide acceptor 6. Coupling of 6 with 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl trichloroacetimidate yielded the trisaccharide 8, and subsequent deallylation and trichloroacetimidation gave 2,3,4-tri-O-benzoyl-β-D-xylopyranosyl-(1→2)-[2-O-acetyl-3,4,6-tri- O-benzoyl-α-D-mannopyranosyl-(1→3)]-4,6-di-O-benzoyl-α-D- mannopyranosyl trichloroacetimidate (9). Condensation of the trisaccharide donor 9 with the disaccharide acceptor 6 gave the pentasaccharide 10 whose 2-O-deacetylation gave the acceptor 11. Glycosylation of 11 with methyl 2,3,4-tri-O-acetyl-α-D-glucopyranosyluronate trichloroacetimidate and subsequent deprotection gave the target hexasaccharide.

Full text of DOI:10.1016/S0008-6215(03)00264-7

Carbohydrate Research 338 (2003) 1719ꢀ1725
/
www.elsevier.com/locate/carres
Synthesis of a hexasaccharide, the repeating unit of O-deacetylated
GXM of C. neoformans serotype A
Jianjun Zhang, Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Academia Sinica, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, PR China
Received 25 March 2003; accepted 3 June 2003
Abstract
b-
the exopolysaccharide from Cryptococcus neoformans serovar A, was synthesized as its allyl glycoside. Thus, 3-O-selective
acetylation of allyl 4,6-O-benzylidene-a- -mannopyranoside afforded 2, and subsequent glycosylation of 2 with 2,3,4-tri-O-
benzoyl- 2)-linked disaccharide 4. Debenzylidenation followed by
-xylopyranosyl trichloroacetimidate furnished the b-(10
benzoylation gave allyl 2,3,4-tri-O-benzoyl-b- -xylopyranosyl-(102)-3-O-acetyl-4,6-di-O-benzoyl-a- -mannopyranoside (5), and
selective 3-O-deacetylation gave the disaccharide acceptor 6. Coupling of 6 with 2-O-acetyl-3,4,6-tri-O-benzoyl-a- -mannopyr-
anosyl trichloroacetimidate yielded the trisaccharide 8, and subsequent deallylation and trichloroacetimidation gave 2,3,4-tri-O-
benzoyl-b- -xylopyranosyl-(102)-[2-O-acetyl-3,4,6-tri-O-benzoyl-a- -mannopyranosyl-(103)]-4,6-di-O-benzoyl-a- -mannopyr-
anosyl trichloroacetimidate (9). Condensation of the trisaccharide donor 9 with the disaccharide acceptor 6 gave the pentasaccharide
D
-GlcpA-(10
/
2)-a-
D
-Manp-(10
/
3)-[b-
D
-Xylp-(10
/
2)]-a-
D
-Manp-(10
/
3)[-b-
D
-Xylp-(10
/
2)]-a-D-Manp, the repeating unit of
D
D
/
D
/
D
D
D
/
D
/
D
10 whose 2-O-deacetylation gave the acceptor 11. Glycosylation of 11 with methyl 2,3,4-tri-O-acetyl-a-
trichloroacetimidate and subsequent deprotection gave the target hexasaccharide.
# 2003 Elsevier Ltd. All rights reserved.
D-glucopyranosyluronate
Keywords: Mannose; Xylose; Glucouronic acid
1. Introduction
enhances HIV infection in human lymphocytes7 and
promotes L-selectin shedding from neutrophils.8 Be-
cause of its prominent virulence, synthesis of the GXM
repeating unit will be very helpful for the research on the
Cryptococcus neoformans, a primary cause of opportu-
nistic infections associated with AIDS, produces glucur-
onoxylomannan (GXM) as the major capsule
component.1,2 There are four major serotypes3 for
structureꢀactivity relationships of oligosaccharides.
/
The synthesis of tri- and tetrasaccharide fragments
corresponding to structures in the capsular polysacchar-
ides of C. neoformans has been reported,9 and the
synthesis of a pentasaccharide, the repeating unit of the
polysaccharide in C. neoformans serovar D, has ap-
peared.10 We have reported in a preliminary commu-
nication11 the successful synthesis of the hexasaccharide
repeating unit of O-deacetylated GXM of C. neofor-
mans serotype A with 4,6-O-isopropylidenated mannose
derivatives as the acceptors based on our previous
studies on the syntheses of cell-wall components. We
now report an alternative highly efficient and conver-
GXM designated Aꢀ
composed of a linear a-(10
/
D (Fig. 1). All four serotypes are
3)-linked mannosyl back-
/
bone with b-glucopyranosyluronic acid, b-xylopyrano-
syl, and 6-O-acetyl substituents.4
GXM is antipathogocytic and poorly immunogenic,
and acapsular strains have significantly reduced viru-
lence.5 In vitro, GXM inhibits leukocyte migration,6
* Corresponding author. Tel.: ꢁ
10-62923563.
E-mail address: fzkong@mail.rcees.ac.cn (F. Kong).
/
86-10-62936613; fax: ꢁ86-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00264-7

1720
J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ1725
/
Fig. 1. Model structures of deacetylated GXM of C. neoformans serotypes AꢀD.
/
1
gent synthesis of the hexasaccharide repeating unit of O-
deacetylated GXM of C. neoformans serotype A.
The H NMR spectrum of 2 showed a characteristic
downfield doublet of doublets at d 5.36 ppm with J2,3
3.2 and J3,4 10.0 Hz for H-3, confirming the 3-OH
ꢂ
/
ꢂ
/
regioselectivity. This selective 3-O-acetylation was the
key step in the synthesis since, on the one hand, 2
contained a free hydroxyl group at the position where
the xylosyl residue should be attached, and on the
another hand, the coupling of 2 with perbenzoylated
xylosyl trichloroacetimidate (3)13 gave a product that
was readily transformed to either an acceptor or a donor
for further reactions. Thus, condensation of 2 with 3
2. Results and discussion
As outlined in Scheme 1, selective acetylation of allyl
4,6-O-benzylidene-a-
tyl chloride in pyridine went smoothly giving allyl 3-O-
acetyl-4,6-O-isopropylidene-a- -mannopyranoside (2)
in high yield (91%), and no acetyl migration was found.
D
-mannopyranoside (1)12 with ace-
D
Scheme 1. (a) CH3COCl, CH2Cl2, Pyridine; (b) TMSOTf, CH2Cl2, ꢃ
Pyridine; (d) 2% CH3COClꢀCH3OH, 0 8C to room temperature; (e) PdCl2, 90% acetic acidꢀ
Cl3CN, DBU, CH2Cl2 2 h; (f) saturated NH3ꢀMeOH, room temperature, 36 h; then H2O, room temperature, 5 h.
/
10 8C to room temperature (dry); (c) 90% TFA; then BzClꢀ
/
/
/
NaOAc, room temperature, 12 h; then
/

J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ
/1725
1721
afforded (10
/
2)-linked disaccharide 4 in good yield
3. Experimental
3.1. General methods
(85%). Because of the presence of a benzylidene group,
selective deacetylation of 4 with 2% CH3COClꢀ
/
MeOH14 was not successful since a benzylidene group
is more sensitive to acidic conditions than either acetyl
or benzoyl groups. Thus, debenzylidenation of 4 was
carried out with 90% trifluoroacetic acid (TFA), and
subsequent benzoylation gave disaccharide 5 (87%).
Melting points were determined with a ‘Mel-Temp’
apparatus. Optical rotations were determined with a
PerkinꢀElmer model 241-MC automatic polarimeter for
/
solutions in a 1-dm jacketed cell. 1H and 13C NMR
spectra were recorded with Varian XL-400 and Varian
XL-200 spectrometers, for solutions in CDCl3 or in D2O
as indicated. Chemical shifts are expressed in ppm
downfield from the Me4Si absorption. Mass spectra
were recorded with a VG PLATFORM mass spectro-
meter using the electronspray-ionization (ESI) mode.
Thin-layer chromatography (TLC) was performed on
silica gel HF with detection by charring with 30% (v/v)
H2SO4 in CH3OH or by UV detection. Column
chromatography was conducted by elution of a column
Selective deacetylation of 5 with 2% CH3COClꢀ
MeOH gave the disaccharide acceptor 6 (73%).
/
Due to the unstability of methyl ester linkage of a
glucuronate residue under either basic or acidic condi-
tions, assembly of the glucuronate unit was arranged at
the end of the reaction series. So, 2-O-acetyl-3,4,6-tri-O-
benzoyl-a-
D
-mannopyranosyl trichloroacetimidate (7)15
was used as the donor to couple 6, and trisaccharide 8
was obtained in satisfactory yield (88%). Subsequent 1-
O-deallylation with PdCl2 and activation with trichlor-
oacetonitrile16a in the presence of potassium carbonate
gave the trisaccharide donor 9 (84%). Again, glycosyla-
tion of 6 with 9 readily afforded the pentasaccharide 10
(8ꢄ
/
100, 16ꢄ
/
240, 18ꢄ
/
300, 35ꢄ/400 mm) of silica gel
(100ꢀ
/
200 mesh) with EtOAcꢀ
/
petroleum ether (bp 60ꢀ
/
90 8C) as the eluent. Analytical LC was performed with
a Gilson HPLC consisting of a pump (model 306),
stainless steel column packed with silica gel (Spherisorb
(83%). Selective deacetylation of 10 with 2% CH3COClꢀ
/
MeOH was accompanied by some decomposition,
perhaps caused by breaking of the xylosyl linkage,
giving the pentasaccharide acceptor 11 in 65% yield.
SiO2, 10ꢄ
ometer (132-RI Detector), and a UVꢀ
(model 118). EtOAcꢀpetroleum ether (bp 60ꢀ
was used as the eluent at a flow rate of 1ꢀ4 mL/min.
Solutions were concentrated at a temperature B60 8C
/
300 or 4.6ꢄ
/
250 mm), a differential refract-
Vis detector
90 8C)
/
/
/
Coupling of 11 with methyl 2,3,4-tri-O-acetyl-a-
D-
/
glucopyranosyluronate trichloroacetimidate (12)16 went
smoothly affording the protected hexasaccharide in
/
under diminished pressure.
good yield (86%). The H and 13C NMR spectra of 13
1
showed a methyl signal (d 3.69 ppm), 13 benzoyl Cꢀ
signals (d 165.9, 165.9, 165.8, 165.4, 165.4, 165.3, 165.3,
165.2, 165.1, 165.0, 164.8, 164.6, 164.6 ppm), four Cꢀ
signals (d 169.0, 168.5, 168.5, 168.3 ppm), and six
anomeric C signals (100.9, JC1,H1 176 Hz, Manp;
100.1, JC1,H1 162 Hz, GluAp; 99.9, JC1,H1 164 Hz,
Xylp; 99.5 JC1,H1 164 Hz, Xylp; 96.7, JC1,H1 173 Hz,
Manp; 96.2, JC1,H1 175 Hz, Manp). It is noted that
/
O
3.2. Allyl 3-O-acetyl-4,6-O-benzylidene-a-D-
mannopyranoside (2)
/O
Compound 1 (3.08 g, 10 mmol) was dissolved in dry
CH2Cl2 (40 mL) containing pyridine (8.1 mL, 100
mmol), then under N2 protection and stirring, a solution
of acetyl chloride (0.8 mL, 11 mmol) in anhyd CH2Cl2
(10 mL) was added dropwise within 30 min at 0 8C. The
reaction mixture was slowly raised to room temperature
(rt) and stirred for 2 h, at the end of which time TLC
ꢂ
/
ꢂ
/
ꢂ
ꢂ
/
ꢂ
/
/
ꢂ
/
although it was difficult to assign the anomeric config-
uration of the two Xylp just from the relatively small
JH1,H2 values (6.1 and 5.2 Hz, respectively), the chemical
shifts of H-1 (d 4.93 and 4.74, respectively) and the
JC1,H1 values clearly indicate the b linkages. Deprotec-
tion of 13 was carried out in a saturated solution of
ammonia in methanol for 36 h, then water (2 equiv) was
added to cleave the methyl ester. After standing at room
temperature for 5 h, the reaction mixture was concen-
trated and purified on a Bio-Gel P2 column (eluent:-
water), affording the target hexasaccharide 14 as a
foamy solid.
In summary, an alternative highly efficient and
convergent synthesis of the hexasaccharide repeat unit
of O-deacetylated GXM of C. neoformans serotype A
was achieved. The strategy presented here also provides
a route to the synthesis of more complex repeating units
of GXM of C. neoformans serotypes B and C.
(3:1 petroleum etherꢀEtOAc) indicated that the reaction
/
was complete. The reaction mixture was diluted with
CH2Cl2 (100 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 column (3:1 petroleum etherꢀ
/
EtOAc)
gave compound 2 (3.17 g, 90.6%) as a syrup: [a]D
ꢁ
/
1
42.08 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
7.46ꢀ7.34 (m, 5 H, PhH), 5.88 (m, 1 H, CH2ꢀ
CHCH2O), 5.55 (s, 1 H, PhCHO2), 5.36 (dd, 1 H, J2,3
3.2, J3,4 10.0 Hz, H-3), 5.20 (m, 1 H, CH2ꢀCHCH2O),
5.23 (m, 1 H, CH2ꢀCHCH2O), 4.89 (d, 1 H, J1,2 1.5 Hz,
H-1), 4.28 (dd, 1 H, J 4.8, 10.6 Hz, H-6a), 4.19 (m, 1 H,
CH2ꢀCHCH2O), 4.15 (dd, 1 H, J1,2 1.5, J2,3 3.2 Hz, H-
2), 4.09 (dd, 1 H, J 10.0, 10.6 Hz, H-6b), 4.02 (m, 1 H,
CH2ꢀCHCH2O), 3.99 (ddd, 1 H, J 4.8, 10.0, 10.6 Hz,
/
/
/
/
/
/

1722
J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ1725
/
H-5), 3.84 (dd, 1 H, J3,4
H, CH3CO). Anal. Calcd for C18H22O7: C, 61.70; H,
ꢂ
/
J4,5 10.0 Hz, H-4), 2.12 (s, 3
CHCH2O), 4.97 (d, 1 H, J1?,2? 3.7 Hz, H-1?), 4.94 (d, 1 H,
J1,2 1.4 Hz, H-1), 4.61 (dd, 1 H, J 3.2, 12.5 Hz, H-6a),
6.33. Found: C, 61.94; H, 6.21.
4.34 (dd, 1 H, J4?,5a? 3.0, J5?a,5?b 11.8 Hz, H-5?a), 4.26 (dd,
1 H, J1,2 1.4, J2,3 3.3 Hz, H-2), 4.22ꢀ
/
4.16 (m, 2 H), 4.08ꢀ
/
3.3. Allyl 2,3,4-tri-O-benzoyl-b-
3-O-acetyl-4,6-O-benzylidene-a-
D
-xylopyranosyl-(10
/
2)-
3.97 (m, 2 H), 3.78 (dd, 1 H, J 4.8, 12.5 Hz, H-6b), 1.97
(s, 3 H, CH3CO). Anal. Calcd for C51H46O16: C, 66.95;
H, 5.07. Found: C, 67.09; H, 5.31.
D
-mannopyranoside (4)
To a cooled solution (ꢃ10 8C) of 2 (3.50 g, 10.0 mmol)
/
and 3 (6.70 g, 11.0 mmol) in anhyd CH2Cl2 (50 mL) was
added TMSOTf (18 mL, 0.1 mmol). The mixture was
stirred for 2 h and then quenched with Et3N (four
drops). The solvents were evaporated in vacuo to give a
residue, which was purified by silica gel column
3.5. Allyl 2,3,4-tri-O-benzoyl-b-
4,6-di-O-benzoyl-a-D-mannopyranoside (6)
D
-xylopyranosyl-(10
/
2)-
To a solution of 5 (4.57 g, 5 mmol) in anhyd CH2Cl2 (10
mL) was added anhyd MeOH (40 mL). Acetyl chloride
(1.0 mL) was then added to the reaction mixture at 0 8C.
The solution was stoppered in a flask and stirred at rt
chromatography (3:1 petroleum etherꢀ/EtOAc) to give
disaccharide 4 (6.76 g, 85.1%) as a foamy solid: [a]D
ꢃ
/
1
38.98 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): d
8.13ꢀ7.33 (m, 20 H, 4 PhH), 5.85 (m, 1 H, CH2ꢀ
CHCH2O), 5.71 (dd, 1 H, J2?,3?ꢂ 3?,4?ꢂ5.8 Hz, H-3?),
until TLC (3:1 petroleum etherꢀEtOAc) showed that
/
/
/
the starting material had disappeared. The solution was
neutralized with Et3N, then concentrated to dryness.
The residue was passed through a short silica gel column
/J
/
5.37 (dd, 1 H, J1?,2? 4.2, J2?,3? 5.8 Hz, H-2?), 5.36 (dd, 1 H,
J2,3 3.5, J3,4 10.5 Hz, H-3), 5.28 (m, 1 H, H-4?), 5.25 (m,
to give 6 (3.20 g, 73.4%) as a foamy solid: [a]D
ꢃ
/
7.58 (c
1.0, CHCl3); H NMR (400 MHz, CDCl3): d 8.05ꢀ7.30
(m, 25 H, 5 PhH), 5.88 (m, 1 H, CH2ꢀCHCH2O), 5.77
(dd, 1 H, J2?,3?ꢂ 3?,4?ꢂ6.9 Hz, H-3?), 5.46 (dd, 1 H,
J3,4 J4,5 9.8 Hz, H-4), 5.37 (dd, 1 H, J1?,2? 5.2, J2?,3?
6.9 Hz, H-2?), 5.29 (m, 1 H, H-4?), 5.19ꢀ5.13 (m, 2 H,
CH2ꢀCHCH2O), 4.97 (d, 1 H, J1?,2? 5.2 Hz, H-1?), 4.94
1
1 H, CH2ꢀ
/
CHCH2O), 5.24 (s, 1 H, PhCHO2), 5.20 (m,
CHCH2O), 4.91 (d, 1 H, J1?,2? 4.2 Hz, H-1?),
/
1 H, CH2ꢀ
/
/
4.80 (d, 1 H, J1,2 1.5 Hz, H-1), 4.59 (dd, 1 H, J 3.5, 12.4
Hz, H-6a), 4.30 (dd, 1 H, J1,2 1.5, J2,3 3.5 Hz, H-2), 4.10
/J
/
ꢂ
/
ꢂ
/
(m, 1 H, CH2ꢀ
J5?a,5?b 10.3 Hz, H-5?a), 3.98 (dd, 1 H, J4?,5b?ꢂ
10.3 Hz, H-5?b), 3.92 (m, 1 H, CH2ꢀCHCH2O), 3.88ꢀ
J4,5 10.5
/CHCH2O), 4.06 (dd, 1 H, J4?,5a? 4.8,
/
/
J5?a,5?b
/
/
/
(d, 1 H, J1,2 0.8 Hz, H-1), 4.61 (dd, 1 H, J 4.0, 12.3 Hz,
H-6a), 4.34 (dd, 1 H, J4?,5a? 2.5, J5?a,5?b 11.9 Hz, H-5?a),
4.24 (dd, 1 H, J4?,5b? 5.8, J5?a,5?b 11.9 Hz, H-5?b), 4.18ꢀ
3.76 (m, 2 H, H-5, H-6b), 3.49 (dd, 1 H, J3,4
ꢂ
/
ꢂ
/
Hz, H-4), 2.14 (s, 3 H, CH3CO); 13C NMR (100 MHz,
CDCl3): d 170.2 (CH3CO), 165.6, 165.1, 165.0 (3 C, 3
/
4.12 (m, 4 H), 3.95ꢀ3.84 (m, 2 H), 1.60 (bs, 1 H, OH).
/
PhCO), 118.1 (CH2ꢀ
96.9 (2 C, 2 C-1), 75.8, 75.3, 69.5, 69.1, 68.6, 68.5, 68.3,
68.2, 64.0, 60.2 (10 C, C-2ꢀ6, C-2?ꢀ CHCH2O),
21.1 (CH3CO). Anal. Calcd for C44H42O14: C, 66.49; H,
5.33. Found: C, 66.20; H, 5.48.
/
CHCH2O), 101.6 (PhCHO2), 98.2,
Anal. Calcd for C49H44O15: C, 67.42; H, 5.08. Found: C,
67.19; H, 5.01.
/
/
5?, CH2ꢀ
/
3.6. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(103)-[2,3,4-tri-O-benzoyl-b- -xylopyran-
osyl-(102)]-4,6-di-O-benzoyl-a- -mannopyranoside (8)
D-manno-
/
D
/
D
3.4. Allyl 2,3,4-tri-O-benzoyl-b-
3-O-acetyl-4,6-di-O-benzoyl-a-
D
-xylopyranosyl-(10
/
2)-
D-mannopyranoside (5)
Compound 6 (2.62 g, 3.0 mmol) and 2-O-acetyl-3,4,6-
tri-O-benzoyl-a- -mannopyranosyl trichloroacetimi-
D
Compound 4 (7.94 g, 10 mmol) was dissolved in 80 mL
90% TFA and stirred for 2 h, at the end of which time
the reaction mixture was poured into toluene (300 mL)
and concentrated. After drying under high vacuum for 2
h, the residue was dissolved in Py (25 mL), and BzCl
(4.70 mL, 40 mmol) was added. The mixture was stirred
at rt for 12 h, then quenched with MeOH (3 mL). The
reaction mixture was evaporated and coevaporated with
toluene in vacuo to give a residue. Purification of the
residue by silica gel column chromatography (3:1
date (7) (2.23 g, 3.3 mmol) were dried together under
high vacuum for 2 h, then dissolved in anhyd CH2Cl2
(50 mL). TMSOTf (18 mL, 0.1 mmol) was added
dropwise at ꢃ10 8C with nitrogen protection. The
/
reaction mixture was stirred for 3 h, during which time
the mixture was allowed to gradually warm to ambient
temperature. Then the mixture was neutralized with
Et3N and concentrated to dryness. Purification of the
residue by column chromatography (1:1 petroleum
etherꢀ
45.68 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): d
8.12ꢀ7.13 (m, 40 H, 8 PhH), 5.89 (dd, 1 H, J2,3 3.3, J3,4
10.0 Hz, H-3 of Manp), 5.77 (dd, 1 H, J3,4 J4,5 9.9
Hz, H-4 of Manp), 5.76 (dd, 1 H, J3,4 J4,5 10.0 Hz,
H-4 of Manp), 5.71 (m, 1 H, CH2ꢀCHCH2O), 5.65 (dd,
1 H, J2,3 J3,4 4.6 Hz, H-3 of Xylp), 5.47 (m, 1 H, H-4
of Xylp), 5.38 (dd, 1 H, J1,2 J2,3 4.6 Hz, H-2 of
/EtOAc) gave 8 (3.66 g, 87.9%) as a syrup: [a]D
petroleum etherꢀ
foamy solid: [a]D
MHz, CDCl3): d 8.12ꢀ
H, CH2ꢀCHCH2O), 5.71 (dd, 1 H, J2?,3?ꢂ
Hz, H-3?), 5.67 (dd, 1 H, J3,4 J4,5 10.0 Hz, H-4), 5.50
(dd, 1 H, J2,3 3.3, J3,4 10.0 Hz, H-3), 5.37 (dd, 1 H, J1?,2?
3.7, J2?,3? 5.0 Hz, H-2?), 5.28ꢀ5.16 (m, 3 H, H-4, CH2ꢀ
/
EtOAc) gave 5 (7.93 g, 86.8%) as a
46.18 (c 1.0, CHCl3); 1H NMR (400
7.29 (m, 25 H, 5 PhH), 5.91 (m, 1
3?,4?ꢂ5.0
ꢃ
/
ꢃ
/
/
/
ꢂ
/
ꢂ
/
/
/
J
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
/
ꢂ
/
ꢂ
/
/
/
ꢂ
/
ꢂ
/

J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ
/1725
1723
Xylp), 5.20 (dd, 1 H, J1,2 1.4, J2,3 3.3 Hz, H-2 of Manp),
5.18 (d, 1 H, J1,2 1.4 Hz, H-1 of Manp), 5.18ꢀ5.10 (m, 2
H, CH2ꢀCHCH2O), 5.07 (d, 1 H, J1,2 4.6 Hz, H-1 of
Xylp), 4.92 (d, 1 H, J1,2 0.8 Hz, H-1 of Manp), 4.85ꢀ
4.76 (m, 2 H), 4.58ꢀ4.54 (m, 2 H), 4.40 (dd, 1 H, J2,3 3.1,
J3,4 9.9 Hz, H-3 of Manp), 4.28 (m, 1 H), 4.24 (dd, 1 H,
J1,2 0.8, J2,3 3.1 Hz, H-2 of Manp), 4.09ꢀ4.03 (m, 2 H),
3.92ꢀ
3.86 (m, 2 H), 1.92 (s, 3 H, CH3CO); 13C NMR
C77H64Cl3NO24: C 61.91; H 4.32. Found: C 61.65; H
4.58.
/
/
/
3.8. Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-a-
pyranosyl-(103)-[2,3,4-tri-O-benzoyl-b- -xylopyran-
osyl-(102)]-4,6-di-O-benzoyl-a- -mannopyranosyl-(10
3)-[2,3,4-tri-O-benzoyl-b- -xylopyranosyl-(102)]-4,6-di-
O-benzoyl-a- -mannopyranoside (10)
D-manno-
/
/
D
/
D
/
/
D
/
/
D
(100 MHz, CDCl3): d 168.7 (CH3CO), 166.1, 165.9,
165.7, 165.3, 165.2, 165.1, 165.0, 164.8 (8 C, 8 PhCO),
Compound 9 (1.64 g, 1.1 mmol) and 6 (873 mg, 1.0
mmol) were coupled under the same conditions as those
used for the preparation of 8 from 7 and 6, giving 10
118.1 (CH2ꢀ
69.7, 69.7, 69.5, 69.3, 69.2, 69.2, 68.9, 68.9, 68.7, 68.5,
68.5, 68.0, 67.7, 63.8, 63.6, 60.3 (15 C, C-2ꢀ6 of Manp;
C-2ꢀ5 of Xylp; CH2ꢀCHCH2O), 20.3 (CH3CO); Anal.
/
CHCH2O), 99.8, 98.6, 96.5 (3 C, 3 C-1),
/
(1.82 g, 82.7%) as a foamy solid: [a]D
ꢃ
/
20.38 (c 1.0,
CHCl3); H NMR (CDCl3, 400 MHz): d 8.10ꢀ7.22 (m,
65 H, 13 PhH), 5.95 (dd, 1 H, J3,4 J4,5 10.0 Hz, H-4
of Manp), 5.79ꢀ5.70 (m, 3 H), 5.62 (dd, 1 H, J3,4
10.0 Hz, H-4 of Manp), 5.57 (dd, 1 H, J3,4
9.9 Hz, H-4 of Manp), 5.49ꢀ5.43 (m, 3 H),
5.28 (m, 2 H), 5.20 (m, 1 H, OCH2CHꢀCH2),
5.17 (d, 1 H, J1,2 5.3 Hz, H-1 of Xylp), 5.17 (m, 1 H,
OCH2CHꢀCH2), 5.13 (s, 1 H, H-1 of Manp), 5.00 (s, 1
1
/
/
/
Calcd for C78H68O24: C, 67.43; H, 4.93. Found: C,
67.26; H, 5.10.
ꢂ
/
ꢂ
/
/
ꢂ
/
J4,5
J4,5
ꢂ
/
ꢂ
/
ꢂ
/
/
3.7. 2-O-Acetyl-3,4,6-tri-O-benzoyl-a-
osyl-(103)-[2,3,4-tri-O-benzoyl-b- -xylopyranosyl-(10
2)]-4,6-di-O-benzoyl-a- -mannopyranosyl
trichloroacetimidate (9)
D-mannopyran-
5.33ꢀ
/
/
/
D
/
D
/
H, H-1 of Manp), 4.78 (d, 1 H, J1,2 4.9 Hz, H-1 of
Xylp), 4.71 (s, 1 H, H-1 of Manp), 1.90 (s, 3 H,
COCH3); 13C NMR (100 MHz, CDCl3): 168.9
(COCH3), 166.0, 165.9, 165.8, 165.4, 165.4, 165.3,
165.2, 165.1, 165.0, 164.8, 164.7, 164.6, 164.5 (13 C, 13
To a solution of 8 (2.08 g, 1.5 mmol) in 90% AcOH (15
mL) containing AcONa (0.44 g, 4.5 mmol) was added
PdCl2 (81 mg, 0.75 mmol), and the mixture was stirred
for 12 h, at the end of which time TLC (2:1 petroleum
COPh), 118.2 (OCH2CHꢀ
96.7 (5 C, 5 C-1), 20.3 (COCH3). Anal. Calcd for
124H106O38: C, 67.56; H, 4.85. Found: C, 67.75; H,
/CH2), 100.0, 99.7, 99.7, 98.9,
etherꢀEtOAc) indicated that the reaction was complete.
/
The mixture was diluted with CH2Cl2 (150 mL), washed
with water and satd aq sodium bicarbonate. The organic
layer was concentrated, and the residue was passed
through a short silica gel column with 1:1 petroleum
C
5.03.
3.9. Allyl 3,4,6-tri-O-benzoyl-a-
D
-mannopyranosyl-(10
2)]-4,6-di-
3)-[2,3,4-tri-O-
2)]-4,6-di-O-benzoyl-a-
/
etherꢀ
3,4,6-tri-O-benzoyl-a-
tri-O-benzoyl-b- -xylopyranosyl-(10
zoyl-a,b- -mannopyranose as a syrup. After drying
/
EtOAc as the eluent to give crude 2-O-acetyl-
-mannopyranosyl-(103)-[2,3,4-
2)]-4,6-di-O-ben-
3)-[2,3,4-tri-O-benzoyl-b-
O-benzoyl-a- -mannopyranosyl-(10
benzoyl-b-
-xylopyranosyl-(10
D-mannopyranoside (11)
D-xylopyranosyl-(10
/
D
/
D
/
D
/
D
/
D
under high vacuum for 2 h, the solid was dissolved in
CH2Cl2 (10 mL), and CCl3CN (0.5 mL, 5 mmol) and
DBU (40 mL, 0.3 mmol) were added. The reaction
mixture was stirred for 2 h, at the end of which time
To a solution of 10 (1.10 g, 0.05 mmol) in anhyd CH2Cl2
(10 mL) was added anhyd MeOH (40 mL). Acetyl
chloride (1.0 mL) was added to the reaction mixture at
0 8C. The solution was stoppered in a flask and stirred at
TLC (2:1 petroleum etherꢀEtOAc) indicated that the
/
reaction was complete. Concentration of the reaction
mixture, followed by purification of the crude product
rt until TLC (3:1 petroleum etherꢀEtOAc) showed that
/
the starting material had disappeared. The solution was
neutralized with Et3N, then concentrated to dryness.
The residue was passed through a short silica gel column
on a silica gel column with 2:1 petroleum etherꢀ
as the eluent, furnished the trisaccharide donor 9 (1.88 g,
83.9%) as a foamy solid: [a]D
42.18 (c 0.8, CHCl3); 1H
NMR (400 MHz, CDCl3): d 8.68 (s, 1 H, CNHCCl3),
8.06ꢀ7.21 (m, 40 H, 8 Ph), 6.43 (s, 1 H, H-1 of Manp),
5.89 (dd, 1 H, J2,3 3.1, J3,4 9.8 Hz, H-3 of Manp), 5.86
(dd, 1 H, J3,4 J4,5 9.8 Hz, H-4 of Manp), 5.76 (dd, 1
H, J3,4 J4,5 9.7 Hz, H-4 of Manp), 5.74 (dd, 1 H,
J1,2 J2,3 4.4 Hz, H-3 of Xylp), 5.48 (dd, 1 H, J1,2 0.8,
/EtOAc
ꢃ
/
to give 11 (700 mg, 64.7%) as a foamy solid: [a]D ꢃ
19.4o
/
1
(c 1.0, CHCl3); H NMR (CDCl3, 400 MHz): d 8.12ꢀ
7.22 (m, 65 H, 13 PhH), 6.02 (dd, 1 H, J3,4 J4,5 9.9
Hz, H-4 of Manp), 5.77 (m, 1 H, OCH2CH ꢀCH2), 5.76
(dd, J1,2 J2,3 6.9 Hz, H-3 of Xylp), 5.72 (dd, J1,2
J2,3 6.1 Hz, H-3 of Xylp), 5.66 (dd, 1 H, J2,3 3.2, J3,4
9.9 Hz, H-3 of Manp), 5.61 (dd, 1 H, J3,4 J4,5 9.8
Hz, H-4 of Manp), 5.57 (dd, 1 H, J3,4 J4,5 9.9 Hz, H-
4 of Manp), 5.47 (dd, 1 H, J1,2 5.1, J2,3 6.1 Hz, H-2 of
Xylp), 5.43ꢀ5.37 (m, 2 H, 2 H-4 of Xylp), 5.35ꢀ5.29 (m,
2 H, H-3 of Xylp; OCH2CHꢀCH2), 5.23 (s, 1 H, H-1 of
/
/
ꢂ
/
ꢂ
/
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
ꢂ
/
J2,3 3.0 Hz, H-2 of Manp), 5.45 (dd, 1 H, J1,2 4.9, J2,3 4.4
Hz, H-2 of Xylp), 5.27 (m, 1 H, H-4 of Xylp), 5.26 (d, 1
H, J1,2 4.9 Hz, H-1 of Xylp), 5.20 (d, 1 H, J1,2 1.1 Hz, H-
1 of Manp), 1.94 (s, 3 H, CH3CO). Anal. Calcd for
ꢂ
/
ꢂ
/
/
/
/

1724
J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ1725
/
Manp), 5.16 (s, 1 H, OCH2CHꢀ
/
CH2), 5.12 (s, 1 H, H-1
OCH2CH ꢀ
/
CH2), 5.31ꢀ
/
5.17 (m, 2 H, OCH2CHꢀ
/
CH2),
of Manp), 4.82 (d, 1 H, J1,2 5.1 Hz, H-1 of Xylp), 4.70
(d, 1 H, J1,2 1.3 Hz, H-1 of Manp), 4.61 (d, 1 H, J1,2 5.4
Hz, H-1 of Xylp); Anal. Calcd for C122H104O37: C,
67.77; H, 4.85. Found: C, 67.93; H, 4.64.
5.13 (s, 1 H, H-1 of Manp), 4.92 (s, 1 H, H-1 of Manp),
4.70 (d, 1 H, J1,2 7.8 Hz, H-1 of GluAp), 4.65 (d, 1 H,
J1,2 2.0 Hz, H-1 of Manp), 4.42 (d, 1 H, J1,2 8.0 Hz, H-1
of Xylp), 4.40 (d, 1 H, J1,2 8.2 Hz, H-1 of Xylp); 13C
NMR (100 MHz, D2O): d 173.6 (ꢁ
/
COONH4), 118.5
3.10. Allyl (methyl 2,3,4-tri-O-acetyl-b-
osyluronate)-(102)-3,4,6-tri-O-benzoyl-a-
pyranosyl-(103)-[2,3,4-tri-O-benzoyl-b- -xylopyran-
osyl-(102)]-4,6-di-O-benzoyl-a- -mannopyranosyl-(10
3)-[2,3,4-tri-O-benzoyl-b- -xylopyranosyl-(102)]-4,6-di-
O-benzoyl-a- -mannopyranoside (13)
D
-glucopyran-
(CH2ꢀCHCH2O), 103.6, 103.5, 102.7, 100.5, 100.5, 97.5
/
/
D-manno-
(6 C-1), 79.0, 78.5, 78.4, 78.3, 78.3, 76.9, 76.2, 75.8, 75.8,
73.9, 73.4, 73.2, 72.9, 72.9, 70.7, 70.3, 69.5, 69.5, 68.5,
67.2, 67.0, 66.7, 66.5, 65.4, 65.3, 61.5, 60.7, 60.6.
MALDI-TOF MS Calcd for the ammonium salt of 14,
C37H63NO30: 1001.9 [M]. Found: 1001.6 (M); 1006.9
/
D
/
D
/
D
/
D
(M-NHꢁ4 ꢁNaꢁ):
/
To a cooled solution (0 8C) of 11 (648 mg, 0.3 mmol)
and methyl 2,3,4-tri-O-acetyl-a- -glucopyranosyluro-
D
nate trichloroacetimidate (12) (240 mg, 0.5 mmol) in
anhyd CH2Cl2 (10 mL) was added TMSOTf (8 mL, 0.05
mmol). The mixture was stirred at this temperature for 2
h, and then quenched with Et3N (one drop). The
solvents were evaporated in vacuo to give a residue,
which was purified by silica gel column chromatography
Acknowledgements
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).
(1:1 petroleum etherꢀ
(638 mg, 85.9%): [a]D
(CDCl3, 400 MHz): d 8.08ꢀ
6.00 (dd, 1 H, J3,4 J4,5 10.0 Hz, H-4 of Manp), 5.79
(m, 1 H, OCH2CH ꢀCH2), 5.71 (dd, 1 H, J2,3 J3,4
/
EtOAc) to give hexasaccharide 13
1
ꢃ26.98 (c 0.5, CHCl3); H NMR
/
/7.23 (m, 65 H, 13 PhH),
ꢂ
/
ꢂ
/
References
/
ꢂ
/
ꢂ
/
6.9 Hz, H-3 of Xylp), 5.15 (s, 1 H, H-1 of Manp), 4.97
(d, 1 H, J1,2 0.7 Hz, H-1 of Manp), 4.93 (d, 1 H, J1,2 6.1
Hz, H-1 of Xylp), 4.74 (d, 1 H, J1,2 5.2 Hz, H-1 of Xylp),
4.78 (s, 1 H, H-1 of Manp), 4.07 (d, 1 H, J1,2 6.8 Hz, H-1
of GluAp), 3.69 (s, 3 H, COOCH3), 1.96, 1.93, 1.27 (3 s,
9 H, 3 COCH3); 13C NMR (100 MHz, CDCl3): 169.0,
168.5, 168.5, 168.3 (4 C, 3 COCH3, COOMe), 165.9,
165.9, 165.8, 165.4, 165.4, 165.3, 165.3, 165.2, 165.1,
165.0, 164.8, 164.6, 164.6 (13 C, 13 COPh), 118.3
1. Cherniak, R. Top. Med. Mycol. 1988, 2, 40ꢀ
2. Bhattacharjee, A. K.; Bennett, J. E.; Glaudemans, C. P. G.
Rev. Infect. Dis. 1982, 6, 619ꢀ624.
3. Wilson, D. E.; Bennett, J. E.; Bailey, J. W. Proc. Soc. Exp.
Biol. Med. 1968, 127, 820ꢀ827.
4. (a) Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaude-
mans, C. P. J. Carbohydr. Res. 1979, 73, 183ꢀ192;
(b) Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaude-
mans, C. P. J. Carbohydr. Res. 1981, 95, 237ꢀ245;
(c) Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaude-
mans, C. P. J. Carbohydr. Res. 1980, 82, 103ꢀ111;
(d) Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaude-
mans, C. P. J. Mol. Immunol. 1979, 16, 531ꢀ540.
5. Fromtling, R. A.; Shadomy, H. J.; Jacobson, E. S.
Mycopathologia 1982, 79, 23ꢀ29.
6. Dong, Z. M.; Murphy, J. W. Infect. Immun. 1995, 63,
770ꢀ778.
/
54.
/
/
/
/
(OCH2CHꢀ
Manp), 100.1 (C-1, JC1,H1
1, JC1,H1 164 Hz, Xylp), 99.5 (C-1, JC1,H1
Xylp), 96.7 (C-1, JC1,H1 173 Hz, Manp), 96.2 (C-1,
JC1,H1 175 Hz, Manp), 51.9 (COOCH3), 20.5, 20.4,
/
CH2), 100.9 (C-1, JC1,H1
162 Hz, GluAp), 99.9 (C-
164 Hz,
ꢂ
/
176 Hz,
/
ꢂ
/
ꢂ
/
ꢂ
/
/
ꢂ
/
ꢂ
/
/
20.4 (4 C, 4 COCH3). Anal. Calcd for C135H120O46: C,
65.42; H, 4.88. Found: C, 65.61; H, 4.79.
/
7. Pettoello-Mantovani, M. A.; Casadevall, M. A.; Kall-
mann, T. R.; Rubinstein, A.; Goldstein, H. Lancet 1992,
3.11. Allyl (b-
mannopyranosyl-(10
mannopyranosyl-(10
D
-glucopyranosyluronic acid)-(10
/
2)-a-D-
2)]-a-
2)]-a-
/3)-[b-
D
-xylopyranosyl-(10
/
D
-
339, 21ꢀ
8. Dong, Z. M.; Murphy, J. W. J. Clin. Invest. 1996, 97,
689ꢀ698.
9. (a) Garegg, P. J.; Olsson, L.; Oscarson, S. J. Carbohydr.
Chem. 1993, 12, 195ꢀ203;
(b) Garegg, P. J.; Olsson, L.; Oscarson, S. Bioorg. Med.
Chem. 1996, 4, 1867ꢀ1878.
/
23.
/3)-[b-
D
-xylopyranosyl-(10
/
D-
/
mannopyranoside, ammonium salt (14)
/
Hexasaccharide 13 (490 mg, 0.2 mmol) was dissolved in
a satd methanolic ammonia (50 mL). After 36 h at rt,
water (1.0 mL) was added to the mixture to cleave the
methyl ester. After stirring at rt for 5 h, the reaction
mixture was concentrated and purified on a Bio-Gel P2
column (eluent:water), affording the target hexasacchar-
/
10. Zegelaar-Jaarsveld, K.; Smits, S. A. W.; van der Marel, G.
A.; van Boom, J. H. Bioorg. Med. Chem. 1996, 4,
1819.
11. Zhang, J.; Kong, F. Tetrahedron Lett. 1839, 2003, 1850.
12. Zhang, J.; Kong, F. Tetrahedron: Asymmetry 2002, 13,
ide 14 (126 mg, 63.0%) as a foamy solid: [a]D
1
0.5, H2O); H NMR (D2O, 400 MHz): d 5.87 (m, 1 H,
ꢁ
/
72.88 (c
243ꢀ252.
/

J. Zhang, F. Kong / Carbohydrate Research 338 (2003) 1719ꢀ
/1725
1725
13. Chen, L.; Kong, F. Carbohydr. Res. 2002, 337, 2335ꢀ
/
15. Zhu, Y.; Kong, F. Synlett 2000, 1783ꢀ
16. (a) Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem.
Biochem. 1994, 50, 21ꢀ125;
(b) Chen, L.; Kong, F. Carbohydr. Res. 2002, 337, 1373ꢀ
1380.
/
1787.
2342.
14. Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L.
/
V.; Kochetkov, N. K. Carbohydr. Res. 1983, 124, c8ꢀ
/
/
c10.