An efficient synthesis of a hexasaccharide - The repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

Article abstract of DOI:10.1016/S0040-4039(03)00119-9

A general method for the synthesis of 2-xylose or glucuronic acid branched (1→3)-linked mannose oligosaccharides has been developed. As a typical example, the synthesis of the methyl glycoside of β-D-GlcpA-(1→2)-α-D-Manp-(1→3)-[β-D-Xylp- (1→2)-]α-D-Manp-(

Full text of DOI:10.1016/S0040-4039(03)00119-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1839–1842
An efficient synthesis of a hexasaccharide—the repeating unit of
the exopolysaccharide from Cryptococcus neoformans serovar A
Jianjun Zhang and Fanzuo Kong*
Research Center for Eco-Environmental Sciences, Academia Sinica, PO Box 2871, Beijing 100085, P.R. China
Received 11 November 2002; revised 3 January 2003; accepted 10 January 2003
Abstract—A general method for the synthesis of 2-xylose or glucuronic acid branched (1“3)-linked mannose oligosaccharides has
been developed. As a typical example, the synthesis of the methyl glycoside of b-
2)-]a- -Manp-(1“3)-[b- -Xylp-(1“2)-]a-
serovar A, was achieved in a regio- and stereoselective manner. © 2003 Elsevier Science Ltd. All rights reserved.
D
-GlcpA-(1“2)-a-
D
-Manp-(1“3)-[b-D-Xylp-(1“
D
D
D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans
Cryptococcus neoformans has been a primary cause of
opportunistic infections in patients diagnosed with
AIDS.1 The yeast C. neoformans is a pulmonary patho-
gen and can disseminate to the central nervous system
causing meningoencephalitis.2 C. neoformans produces
large amounts of a polysaccharide composed of man-
nose, xylose, and glucuronic acid. This polysaccharide,
termed glucuronoxylomannan (GXM),3 is the major
constituent of the cryptococcal capsule.4
essential for binding, though they have a significant
contribution.3
Synthesis of the repeating units of the polysaccharide
from C. neoformans serotypes A–D is of interest since it
can afford synthetic samples in sufficient amounts for
studies on the relationship of oligosaccharide structure-
bioactivity. However, this synthesis is not easy because
of the relatively poor reactivity of the 2-axial hydroxy
group of mannose, and the steric hindrance caused by
the 2,3-substitution of the mannose residues. The syn-
thesis of trisaccharide and tetrasaccharide fragments
corresponding to structures in capsular polysaccharides
of C. neoformans has been reported11,12 and the synthe-
sis of a pentasaccharide—the repeating unit of the
GXM occurs as four major serotypes,5 designated A–D
(Scheme 1). All the four serotypes have a linear a-1,3-
linked mannosyl backbone with b-glucopyran-
osyluronic acid and b-xylopyranosyl substituents.6–9 In
addition, the backbone is substituted with variable
amounts of 6-O-acetyl groups, with serotype D being
the most heavily O-acetylated and serotype C the least
O-acetylated.10 However, the O-acetyl groups are not
polysaccharide in C. neoformans serovar
D has
appeared.13 In these syntheses, multiple steps and
orthogonal masking groups were involved making the
Scheme 1. Model structures of GXM of C. neoformans serotypes A–D.
* Corresponding author.
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00119-9

1840
J. Zhang, F. Kong / Tetrahedron Letters 44 (2003) 1839–1842
Scheme 2. Reagents and conditions: (a) (i) THF–CH3OH, 1.5 N NH3, rt, 2–3 h; (ii) CH2Cl2, CCl3CN (2.0 equiv.), K2CO3 (2.0
,
equiv.), rt, 12 h, 71%; (b) TMSOTf (0.01 equiv.), 4 A MS, CH2Cl2, −20°C, 2–4 h (74% for 4, 67% for 8, 88% for 15); (c)
,
Ac2O–pyridine, 100%; (d) CH3OH saturated with ammonia, rt, 12 h, 100%; (e) 10 (2 equiv.), TMSOTf (0.1 equiv.), 4 A MS,
CH2Cl2, −0°C, 0.5 h; then TMSOTf (1.0 equiv.), 0.5 h, and further 10 (2 equiv.) was added, 42%; (f) (i) 90% HOAc, 60°C, 20 h;
(ii) BzCl–pyridine, rt, 10 h, 91%; (g) 2% CH3COCl in CH2Cl2–CH3OH, rt, 20 h, 67%; (h) methanol saturated with ammonia, rt,
36 h, then water (2 equiv.) was added, 5 h, 65%.

J. Zhang, F. Kong / Tetrahedron Letters 44 (2003) 1839–1842
1841
procedure rather complex. As a result, it would be
difficult to synthesize by the reported methods, the
higher oligosaccharides—the repeating units of C.
neoformans serotypes A, B, and C. Previously, we have
reported the regio- and stereoselective synthesis of
oligosaccharides with un- or lightly protected
mannose14 and rhamnose15 as the glycosyl acceptors
and glycosyl trichloroacetimidates as the donors giving
satisfactory results. We present herein, for the first time,
the regio- and stereoselective synthesis of a frame-
shifted hexasaccharide repeating unit16 I (structure 16
in Scheme 2) of O-deacetylated GXM of C. neoformans
serotype A with lightly protected mannose derivatives
as the acceptors.
in 67% yield. Coupling of 13 with methyl 2,3,4-tri-O-
-glucopyranosyluronate trichloroacetimidate
acetyl-a-
D
14 went smoothly affording the protected hexasaccha-
1
ride in good yield (88%). The H NMR spectrum of 15
showed two methyl signals (l 3.69 ppm and 3.23 ppm,
respectively), thirteen benzoyl CꢀO signals (l 165.9,
165.9, 165.9, 165.4, 165.4, 165.4, 165.3, 165.2, 165.1,
165.0, 164.9, 164.6, 164.6 ppm), four CꢀO signals (l
167.0, 168.5, 168.5, 168.3 ppm), six anomeric C signals
(100.9, JC1,H1=175 Hz, Manp; 100.3, JC1,H1=163 Hz,
GluAp; 99.9, JC1,H1=164 Hz, Xylp; 99.5, JC1,H1=163
Hz, Xylp; 98.5, JC1,H1=172 Hz, Manp; 95.2, JC1,H1
=
176 Hz, Manp).20 Deprotection of 15 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 concentrated and purified on a
Bio-Gel P2 column (eluent: water), affording the target
hexasaccharide 16 as a foamy solid.
As shown in Scheme 2, 1,2,3-tri-O-acetyl-4,6-O-iso-
propylidene- -mannopyranose 1, obtained from selec-
D
tive 4,6-O-isopropylidenation of mannose17 with
2-methoxypropene followed by acetylation, was chosen
as the starting material. Selective 1-O-deacetylation
with ammonia in THF–MeOH followed by
trichloroacetimidation18 with trichloroacetonitrile in the
presence of potassium carbonate gave the donor 2,3-
In summary, an efficient synthesis of a hexasaccharide
repeat unit of O-deacetylated GXM of C. neoformans
serotype A with lightly protected mannose derivatives
as the acceptors was achieved. The strategy presented
here also provides a route to the synthesis of more
complex repeating units of GXM of C. neoformans
serotype B and C.
di-O-acetyl-4,6-O-isopropylidene-a-
trichloroacetimidate 2. Condensation of 2 with the
-mannopy-
D-mannopyranosyl
acceptor methyl 4,6-O-isopropylidene-a-
D
ranoside 3 selectively afforded the (1“3)-linked disac-
charide 4 (74%). The regioselectivity of the coupling
1
was confirmed by acetylation of 4 to give 5, and the H
NMR spectrum of
5 showed a newly emerged
Acknowledgements
downfield doublet of doublets at l 5.34 ppm with
1,2=1.5 and J2,3=3.0 Hz for H-2 compared to that of
J
4. Deacetylation of 4 or 5 in a solution of ammonia in
methanol furnished the disaccharide triol acceptor 6
quantitatively. Again, 3-O-selective glycosylation of 6
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).
with the donor 2-O-acetyl-3,4,6-tri-O-benzoyl-a-D-
mannopyranosyl trichloroacetimidate 7 yielded (1“3)-
linked trisaccharide 8 (67%). Confirmation of the
3-regioselectivity was carried out by acetylation of 8 to
give the trisaccharide 9 which showed two characteristic
signals at l 5.40 ppm with J1,2=1.3, J2,3=3.4 Hz for
H-2, and 5.22 ppm with J1,2=1.3, J2,3=2.9 Hz for H-2%,
respectively. The trisaccharide 8 was an ideal acceptor
since it contained two free hydroxyl groups at the
positions where the xylosyl residue should be attached.
Thus, TMSOTf promoted xylosylation of 8 with 2,3,4-
References
1. Dismukes, W. E. J. Infect. Dis. 1988, 157, 624.
2. Diamond, R. D. In Principles of Infectious Diseases;
Mandell, G. L.; Douglas, R. G., Jr.; Bennett, J. E., Eds.;
Churchhill Livingstone: New York, 1990; pp. 1980–1989.
3. Otteson, E. W.; Welch, W. H.; Kozel, T. R. J. Biol.
Chem. 1994, 269, 1858.
tri-O-benzoyl- -xylopyranosyl trichloroacetimidate 10
D
was carried out. It was noted that at the initial stage of
the coupling, formation of the tetrasaccharide interme-
diate was quite fast and little pentasaccharide was
obtained. The quantity of pentasaccharide did not
increase along with the reaction time. For completion
of the reaction, an ‘inverse Schmidt’ strategy was used.
Thus, further TMSOTf was added, and after stirring
the reaction mixture for 20 min, further 2 equiv. of
donor 10 was added giving the pentasaccharide 11 in
fair yield (42%). De-isopropylidenation of 11 in 60%
aqueous acetic acid at 60°C followed by benzoylation
with benzoyl chloride in pyridine gave the protected
pentasaccharide 12. Selective deacetylation of 12 with
2% acetyl chloride–methanol19 was accompanied by
some decomposition, perhaps caused by breaking of
xylosyl linkage, giving the pentasaccharide acceptor 13
4. Cherniak, R.; Reiss, E.; Slodki, M. E.; Plattner, R. D.;
Blumer, S. O. Mol. Immunol. 1980, 17, 1025.
5. Wilson, D. E.; Bennett, J. E.; Bailey, J. W. Proc. Soc.
Exp. Biol. Med. 1968, 127, 820.
6. Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaudemans,
C. P. J. Carbohydr. Res. 1979, 73, 183.
7. Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaudemans,
C. P. J. Carbohydr. Res. 1981, 95, 237.
8. Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaudemans,
C. P. J. Carbohydr. Res. 1980, 82, 103.
9. Bhattacharjee, A. K.; Kwon-Chung, K. J.; Glaudemans,
C. P. J. Mol. Immunol. 1979, 16, 531.
10. Kozel, T. R.; Hermerath, C. A. Infect. Immun. 1984, 43,
879.
11. Garegg, P. J.; Olsson, L.; Oscarson, S. J. Carbohydr.
Chem. 1993, 12, 195.

1842
J. Zhang, F. Kong / Tetrahedron Letters 44 (2003) 1839–1842
12. Garegg, P. J.; Olsson, L.; Oscarson, S. Bioorg. Med.
Chem. 1996, 4, 1867.
1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): l 8.11–7.34
(m, 65 H, 13 PhH), 5.94 (dd, 1 H, J3,4=J4,5=10.0 Hz,
H-4, Manp), 5.77 (dd, 1 H, J2,3=3.2 Hz, J3,4=10.0 Hz,
H-3, Manp), 5.71 (dd, J1,2=J2,3=6.3 Hz, H-2, Xylp),
5.59 (dd, 1 H, J3,4=J4,5=10.0 Hz, H-4, Manp), 5.54 (dd,
1 H, J3,4=J4,5=9.9 Hz, H-4, Manp), 5.47–5.40 (m, 3 H),
5.31–5.27 (m, 2 H), 5.18 (d, 1 H, J1,2=0.7 Hz, H-1,
Manp), 5.16 (dd, 1 H, J1,2=1.0 Hz, J2,3=3.0 Hz, H-2,
Manp), 4.99 (d, 1 H, J1,2=0.8 Hz, H-1, Manp), 4.80 (d,
1 H, J1,2=4.8 Hz, H-1, Xylp), 4.53 (d, 1 H, J1,2=1.0 Hz,
H-1, Manp), 4.45 (d, 1 H, J1,2=6.1 Hz, H-1, Xylp), 3.19
(s, 3 H, OCH3), 1.90 (s, 3 H, COCH3). 13C NMR (100
MHz, CDCI3): 168.9 (COCH3), 166.0, 165.9, 165.8,
165.4, 165.3, 165.3, 165.3, 165.2, 165.1, 165.0, 164.9,
164.7, 164.6 (13 C, 13 COPh), 99.7, 99.7, 99.7, 99.0, 98.5
(5 C, 5 C-1), 54.8 (OCH3), 20.3 (COCH3); Anal. calcd for
C122H104O38: C, 67.27; H, 4.81. Found: C, 67.45; H, 5.96.
13. ZegelaarJaarsveld, K.; Smits, S. A. W.; van der Marel, G.
A.; van Boom, J. H. Bioorg. Med. Chem. 1996, 4, 1819.
14. (a) Zhu, Y.; Kong, F. Synlett 2001, 1217; (b) Zhu, Y.;
Kong, F. Synlett 2000, 663; (c) Zhang, J.; Kong, F.
Tetrahedron: Asymmetry 2002, 13, 243; (d) Zhu, Y.;
Chen, L.; Kong, F. Carbohydr. Res. 2002, 337, 207.
15. (a) Zhang, J.; Kong, F. J. Carbohydr. Chem. 2002, 21, 79;
(b) Zhang, J.; Kong, F. Carbohydr. Res. 2002, 337, 391;
(c) Zhang, J.; Zhu, Y.; Kong, F. Carbohydr. Res. 2001,
336, 329.
16. Sheng, S.; Cherniak, R. Carbohydr. Res. 1997, 301, 33.
17. Copeland, C.; Stick, R. V. Aust. J. Chem. 1978, 31, 1371.
18. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem.
Biochem. 1994, 50, 21–125.
19. Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L.
V.; Kochetkov, N. K. Carbohydr. Res. 1983, 124, c8.
20. All new compounds gave satisfactory elemental analysis
results. Selected physical data for some key compounds
1
For 15: [h]D −22.5 (c 0.5, CHCl3); H NMR (CDCl3, 400
MHz): l 8.13–7.30 (m, 65 H, 13 PhH), 6.00 (dd, 1 H,
J
3,4=J4,5=10.0 Hz, H-4, Manp), 5.71 (dd, 1 H, J2,3=3.2
1
are as follows, for 8: [h]D +91.7 (c 1.0, CHCl3); H NMR
Hz, J3,4=10.0 Hz, H-3, Manp), 5.15 (d, 1 H, J1,2=1.0
Hz, H-1, Manp), 4.96 (d, 1 H, J1,2=0.9 Hz, H-1, Manp),
4.75 (d, 1 H, J1,2=4.9 Hz, H-1, Xylp), 4.60 (d, 1 H,
(CDCl3, 400 MHz): l 7.95–7.34 (m, 15 H, 3 PhH), 5.94
(dd, 1 H, J3,4=J4,5=10.0 Hz, H-4), 5.75 (dd, 1 H,
J2,3=3.3 Hz, J3,4=10.0 Hz, H-3), 5.53 (dd, 1 H, J1,2=1.8
J1,2=4.8 Hz, H-1, Xylp), 4.56 (d, 1 H, J1,2=1.0 Hz, H-1,
Hz, J2,3=3.3 Hz, H-2), 5.43 (d, 1 H, J1,2=1.8 Hz, H-1),
5.31 (d, 1 H, J1,2=1.0 Hz, H-1), 4.71 (d, 1 H, J2,1=1.0
Hz, H-1), 4.66–4.58 (m, 3 H), 4.26–4.18 (m, 4 H), 4.04–
3.99 (m, 2 H), 3.90–3.82 (m, 4 H), 3.68–3.63 (m, 2 H),
3.36 (s, 3 H, OCH3), 2.17 (s, 3 H, COCH3), 1.56, 1.45 (2s,
6 H, isopropylidene), 1.37 (s, 6 H, isopropylidene). 13C
NMR (100 MHz, CDCl3): 169.5 (COCH3), 166.3, 165.6,
163.6 (5 C, 5 COPh), 101.2, 100.8 (2 C, 2 Me2C), 99.9,
99.4, 98.3 (3 C, 3 C-1), 54.8 (OCH3), 29.1, 28.9 (2 C,
CH3CCH3), 20.6 (COCH3), 19.2, 19.0 (2 C, CH3CCH3).
Anal. calcd for C48H56O20: C, 60.50; H, 5.92. Found: C,
60.45; H, 5.66. For 9: [h]D +82.6 (c 1.0, CHCl3); 1H
NMR (CDCl3, 400 MHz): l 8.10–7.23 (m, 15 H, 3 PhH),
6.04 (dd, 1 H, J3,4=J4,5=10.0 Hz, H-4), 5.64 (dd, 1 H,
Manp), 4.04 (d, 1 H, J1,2=6.6 Hz, H-1, GluAp), 3.69 (s,
3 H, COOCH3), 3.23 (s, 3 H, OCH3), 1.96, 1.92, 1.31 (3s,
9 H, 3 COCH3). 13C NMR (100 MHz, CDCl3): 167.0,
168.5, 168.5, 168.3 (3 C, 3 COCH3, COOMe), 165.9,
165.9, 165.9, 165.4, 165.4, 165.4, 165.3, 165.2, 165.1,
165.0, 164.9, 164.6, 164.6 (13 C, 13 COPh), 100.9 (C-1,
J
C1,H1=175 Hz, Manp), 100.3 (C-1, JC1,H1=163 Hz,
GluAp), 99.9 (C-1, JC1,H1=164 Hz, Xylp), 99.5 (C-1,
C1,H1=163 Hz, Xylp), 98.5 (C-1, C1,H1=172 Hz,
J
J
Manp), 95.2 (C-1, JC1,H1=176 Hz, Manp), 54.8 (OCH3),
52.3 (COOCH3), 20.5, 20.3, 20.2 (COCH3); Anal. Calcd
for C133H118O46: C, 65.13; H, 4.85. Found: C, 65.45; H,
4.66. For 16: [h]D +99.6 (c 0.5, H2O); 1H NMR (D2O,
400 MHz): l 5.11 (s, 1 H, H-1, Manp), 4.75 (s, 1 H, H-1,
Manp), 4.31 (s, 1 H, H-1, Manp), 4.29 (d, 1 H, J1,2=8.0
Hz, H-1, GluAp), 4.12 (d, 1 H, J1,2=8.9 Hz, H-1, Xylp),
4.10 (d, 1 H, J1,2=9.2 Hz, H-1, Xylp), 3.33 (s, 3 H,
OCH3); 13C NMR (100 MHz, D2O): 174.1 (ꢁCOONH4),
103.4, 103.3, 103.2, 102.5, 100.3, 100.2 (6 C-1), 79.0, 78.5,
78.4, 78.3, 78.3, 76.4, 76.2, 75.9, 75.8, 73.6, 73.4, 73.2,
72.9, 72.9, 70.7, 70.3, 69.5, 69.5, 68.4, 67.0, 66.7, 66.5,
65.4, 65.3, 61.5, 60.6, 60.6, 56.6 (O-CH3). MALDI–TOF
MS calcd for the ammonium salt of 16, C35H61O30N:
975.8 [M]. found: 975.8 (M); 980.9 (M−NH+4+Na+).
J
2,3=3.2 Hz, J3,4=10.0 Hz, H-3), 5.45 (dd, 1 H, J1,2=2.0
Hz, J2,3=2.9 Hz, H-2), 5.40 (dd, 1 H, J1,2=1.3 Hz,
2,3=3.4 Hz, H-2), 5.32 (d, 1 H, J1,2=1.7 Hz, H-1), 5.22
(dd, 1 H, J1,2=1.3 Hz, J2,3=2.9 Hz, H-2), 5.10 (d, 1 H,
1,2=1.2 Hz, H-1), 4.67 (dd, 1 H, J2,3=2.9 Hz, J3,4=12.1
J
J
Hz, H-3), 4.63 (d, 1 H, J1,2=1.2 Hz, H-1), 4.46–4.38 (m,
2 H), 4.09–4.05 (m, 4 H), 3.87–3.81 (m, 4 H), 3.70–3.60
(m, 2 H), 3.36 (s, 3 H, OCH3), 2.31, 2.18, 2.07 (3s, 9 H,
3 COCH3), 1.56, 1.52 (2s, 6H, isopropylidene), 1.37 (s,
6H, isopropylidene). Anal. calcd for C52H60O22: C, 60.22;
H, 5.83. Found: C, 60.47; H, 5.61. For 12: [h]D −58.6 (c