Synthesis of Cryptococcus neoformans Capsular Polysaccharide Structures. IV. Construction of Thioglycoside Donor Blocks and their Subsequent Assembly

Article abstract of DOI:10.1081/CAR-120026459

Di- and trisaccharide thioglycoside building blocks, ethyl (2,3,4-tri-O-benzyl-β-D-xylopyranosyl)-(1 → 2)-3-O-allyl-4,6-di-O- benzyl-1-thio-α-D-mannopyranoside, ethyl (2,3,4-tri-O-benzyl-β-D- xylopyranosyl)-(1 → 2)-6-O-acetyl-3-O-allyl-4-O-benzyl-1-thio-α

Full text of DOI:10.1081/CAR-120026459

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Synthesis of Cryptococcus neoformans Capsular
Polysaccharide Structures. IV. Construction of
Thioglycoside Donor Blocks and Their Subsequent
Assembly
Mia Alpe a , Stefan Oscarson a & Pär Svahnberg a
a Department of Organic Chemistry , Arrhenius Laboratory , Stockholm University , Floor 6,
Stockholm, S‐106 91, Sweden
Published online: 23 Feb 2007.
To cite this article: Mia Alpe , Stefan Oscarson & Pär Svahnberg (2003) Synthesis of Cryptococcus neoformans Capsular
Polysaccharide Structures. IV. Construction of Thioglycoside Donor Blocks and Their Subsequent Assembly , Journal of
Carbohydrate Chemistry, 22:7-8, 565-577, DOI: 10.1081/CAR-120026459
To link to this article: http://dx.doi.org/10.1081/CAR-120026459
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, Nos. 7 & 8, pp. 565–577, 2003
Synthesis of Cryptococcus neoformans Capsular
Polysaccharide Structures. IV. Construction
of Thioglycoside Donor Blocks and Their
Subsequent Assemblyy
*
Mia Alpe, Stefan Oscarson, and Pa¨r Svahnberg
Department of Organic Chemistry, Arrhenius Laboratory,
Stockholm University, Sweden
ABSTRACT
Di- and trisaccharide thioglycoside building blocks, ethyl (2,3,4-tri-O-benzyl-b-D-
xylopyranosyl)-(1 ! 2)-3-O-allyl-4,6-di-O-benzyl-1-thio-a-D-mannopyranoside,
ethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-6-O-acetyl-3-O-allyl-4-O-ben-
zyl-1-thio-a-D-mannopyranoside and ethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-
(1 ! 4)-[(2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)]-3-O-allyl-6-O-benzyl-1-
thio-a-D-mannopyranoside, corresponding to repetitive structures in the capsular
polysaccharide (CPS) of Cryptococcus neoformans have been synthesised using
silver triflate-promoted couplings between benzobromoxylose and properly pro-
tected mannose ethyl thioglycosides. The blocks contain an orthogonal allyl group
in the 3-position of the mannose residue to allow continued formation of the
(1 ! 3)-linked mannan backbone of the CPS. They have benzyl ethers as persis-
tent protecting groups to facilitate access to the acetylated target structures. As-
sembly of the blocks employing DMTST as promoter in diethyl ether afforded in
high yield and complete stereoselectivity penta- and hexasaccharide motifs from C.
yThis paper is dedicated to Professor Ge´rard Descotes on the occasion of his 70th birthday.
*
Correspondence: Stefan Oscarson, Department of Organic Chemistry, Arrhenius Laboratory
Floor 6, Stockholm University, S-106 91 Stockholm, Sweden; E-mail: s.oscarson@organ.su.se.
565
DOI: 10.1081/CAR-120026459
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

566
Alpe, Oscarson, and Svahnberg
neoformans serotype A–C. The latter were deallylated into new acceptors to allow
synthesis of larger CPS-fragments.
Key Words: Convergent synthesis; Modular approach; Block synthesis;
Glycoconjugate vaccines.
INTRODUCTION
The fungi Cryptococcus neoformans is an opportunistic species causing severe
diseases, i.e., meningitis, and death, especially in immunodeficient patients.[1,2] C.
neoformans is divided into different serotypes, A–D, depending on the structure of the
surface capsular polysaccharide (CPS), which is an important virulence factor.[3–5] The
major polysaccharide is a heteropolymer made of an a-D-(1 ! 3)-mannan backbone
substituted with b-D-xylose residues in the 2 and 4-positions, b-D-glucuronic acid
residues in the 2-positions and acetyl groups in the 6-positions.[6,7] The structures are
more or less repeated in triads (Figure 1) and the different serotypes are defined by the
amount and position of the xylose substituents (Table 1). The acetylation pattern is
believed to be a major immunological determinant[7] but little else is known about the
distribution of the 6-O-acetyl groups.
To investigate in more detail the nature of the immunological determinants and the
biosynthesis of the C. neoformans CPS, well-defined synthetic oligosaccharides cor-
responding to fragments of the native CPS are in demand.
An attractive synthetic pathway to various CPS structures would be to combine di-
and trisaccharide thioglycoside building blocks, corresponding to the substituted
mannose motifs, through the formation of the internal a-(1 ! 3) linkages of the
mannan backbone. With four disaccharide blocks (I, II, IV and V) and two trisac-
charide blocks (III and VI) (Figure 2), all possible structures could theoretically be
constructed. In initial attempts, however, this approach met with severe difficulties,
regarding not only the synthesis of the building blocks but also their coupling.[8,9]
Therefore another approach was employed to manufacture a number of C. neoformans
CPS structures including tetrasaccharides.[10–12] In this, the mannan backbone was built
up first and the xylose, glucuronic acid and acetyl residues were subsequently intro-
duced. The same strategy has recently been used for the synthesis of a type A hex-
asaccharide structure.[13] Although so far successful, the synthesis of larger fragments
Figure 1. Schematic structure of deacetylated C. neoformans CPS.

Synthesis of Cryptococcus neoformans. IV
Table 1. Correlation between Xylp substitution and serotype.
Serotype
567
Xylp pos.
A
B
C
D
2’
4’
4@
+
+
À
+
+
+
+
À
À
À
À
À
using this technique would probably be limited because of the number of rather
complex reactions involved on large structures. Thus, the block approach has therefore
always been pursued. The successful formation and subsequent assembly of xylose-
containing di- and trisaccharide building blocks (corresponding to structures I–III in
Figure 2) are described herein.
RESULTS AND DISCUSSION
First, to allow later formation of immunogenic glycoconjugates, the mannose
acceptor 7 was constructed from the known peracetylated spacer-bearing glycoside
1.[14] Thus, deacetylation of 1 (!2), and subsequent 4,6-O-benzylidene acetal for-
mation(!3), tin activation and regioselective 3-O-p-methoxybenzylation[15] (!4),
debenzylidenation (!5), and benzylation (!6) gave the key acceptor 7 in 27% overall
yield after final removal of the p-methoxybenzyl group (Scheme 1).
Figure 2. Desired thioglycoside building blocks.

568
Alpe, Oscarson, and Svahnberg
Scheme 1. i: NaOMe, MeOH, 99%; ii: PhCH(OMe)2, p-TsOH, MeCN, 70%; iii: a) Bu2SnO,
MeOH, reflux; b) MBnCl, CsF, DMF, 64%; iv: HOAc (aq 70%), 70°C, 85%, v: BnBr, NaH, DMF,
88%; vi: DDQ, CH2Cl2/H2O, 0°C, 82%.
The building blocks were prepared from the thioglycoside derivative 8,[16] which
was regioselectively allylated in the 3-position using tin activation to give 9 (86%)
(Scheme 2). Analogously to earlier syntheses,[10,11] silver triflate-promoted glycosyla-
tion of the latter with benzobromoxylose 10[17] gave the b-linked disaccharide 11 in
excellent yield (ꢀ95%). Since the continued use of acyl protecting groups was pro-
hibited, due to the presence of acetyl substituents in the target compounds, the benzoyl
groups in 11 were changed into benzyl groups by Zemple´n deacylation (!12, 1H
NMR: 4.58 (d, 1H, J = 9 Hz, H-1’)) followed by conventional benzylation (!13, 71%
from 9). This sequence could be performed on a large scale and gram quantities of
disaccharide 13 were efficiently synthesised. To allow for the introduction of a 6-O-
acetyl group and also to release torsional strains in the donor,[18] which could explain
earlier low coupling yields,[8,9] the benzylidene acetal in 13 was opened under re-
ductive conditions that normally yield mainly the 4-O-benzyl derivative (BH3–Me3N,
AlCl3, CH2Cl2/Et2O).[19] Here, however, an inseparable 1:1-mixture of the 4-O- and the
6-O-benzyl derivatives 14a and 14b was obtained. Since both the 6-O-acetylated and
benzylated derivates were desired this was not a disadvantage. Regioselective acety-
lation[20] of the obtained mixture afforded an easily separable mixture of the first
thioglycoside donor 15 (35%, compare I in Figure 2) and 14b. The latter was subse-
quently benzylated to give the donor 16 (compare II in Figure 2) in 34% yield from 13.
Notably, 14b also comprises the possibility to construct a trisaccharide donor block
upon introduction of another xylose residue in the 4-position.
Scheme 2. i: a) Bu2SnO, MeOH, reflux; b) AllBr, CsF, DMF, 86%; ii: AgOTf, DTBP, CH2Cl2,
À40°C; iii: NaOMe, MeOH, 82%; iv: BnBr, NaH, DMF, 87%; v: BH3–Me3N, AlCl3, CH2Cl2/
Et2O, 0°C; vi: AcCl, collidine, CH2Cl2, À70°C; vii: BnBr, NaH, DMF, 91%.

Synthesis of Cryptococcus neoformans. IV
569
Scheme 3. i: NaCNBH3, HCl/Et2O, THF, 75%; ii: 10, AgOTf, DTBP, CH2Cl2, À40°C; iii:
NaOMe, MeOH, 65% over two steps; iv: BnBr, NaH, DMF, 61%.
However, a more efficient route to a trisaccharide building block (compare III in
Figure 2) was to introduce the two xylose moieties at the same time (Scheme 3).
Reductive opening of the benzylidene acetal in 9 yielded the 2,4-diol 17 (75%),
which was glycosylated with donor 10 using silver triflate as promoter to yield the
trisaccharide 18. Subsequent deacylation (!19), and benzylation, then afforded the
desired block 20 in 40% yield from 17.
Scheme 4. i: DMTST, Et2O; ii: PdCl2, EtOH/MeOH.

570
Alpe, Oscarson, and Svahnberg
Assembly of the available blocks was then investigated. Dimethyl(methylthio)-
sulfonium triflate (DMTST)-promoted glycosylations in diethyl ether of acceptor 7 with
disaccharide donors 15 and 16 gave the a-linked trisaccharides 21 and 25, respectively,
in high yields (Scheme 4). Furthermore, after removal of the 3’-O-allyl group with
PdCl2, both the obtained trisaccharide acceptors 22 and 26, could be efficiently elon-
gated by new stereoselective DMTST-promoted glycosylations proving the assembly
concept. Glycosylation of acceptor 22 with donor 16 afforded pentasaccharide 23 (65%,
95% calculated on consumed acceptor), whereas coupling of acceptor 26 with donor 16
or trisaccharide donor 20 yielded pentasaccharide 27 (85%) and hexasaccharide 29
(85%), respectively. Subsequent deallylations then afforded new acceptors, the penta-
saccharides 24 (80%) and 28 (62%) and hexasaccharide 30 (62%), into which glu-
curonic acid-containing building blocks can be introduced to produce structures corres-
ponding to Cryptococcus serotypes A–C.
In conclusion, xylose-containing di- and trisaccharide motifs of the mannan
backbone in the C. neoformans CPS have been efficiently synthesised as thioglycoside
blocks, which were shown to be good donors in glycosylation reactions. This modular
approach allows an iterative selective deprotection/glycosylation scheme to synthesize
larger CPS oligosaccharide structures including acetylated target molecules. Access to
similar glucuronic acid-containing building blocks should allow the synthesis of a large
variety of CPS structures from the different serotypes found in C. neoformans.
EXPERIMENTAL
General methods. TLC was carried out on Merck precoated 60 F254 plates using
UV-light and/or 8% aq sulfuric acid for visualization. Column chromatography was
performed on silica gel (0.040–0.063 mm, Amicon). NMR spectra were recorded in
CDCl3 (internal Me4Si, d = 0.00) at 25°C on a Varian 300 MHz or 400 MHz instru-
ment. MALDI-TOF spectra were recorded on a Bruker Biflex III instrument using
2’,4’,6’-trihydroxyacetophenone trihydrate (THAP) as matrix. Organic phases were dried
over Na2SO4 before evaporation, which was performed under reduced pressure.
2-Azidoethyl 2,4,6-tri-O-benzyl- -D-mannopyranoside (7). A solution of 1[14]
(1.26 g, 3.02 mmol) in MeOH (25 mL) was treated with a catalytic amount of 1 M
methanolic NaOMe at rt overnight. Dowex 50 (H+) ion-exchange resin was added and
the mixture was filtered, and concentrated to give 2-azidoethyl a-D-mannopyrano-
side[21] (2, 742 mg, 99%) as a white solid; NMR data (CDCl3/CD3OD): 13C, d 50.8,
61.3, 66.5, 66.7, 71.0, 71.6, 72.9 and 100.4. a,a-Dimethoxytoluene (536 mL, 3.57 mmol)
and p-toluenesulfonic acid (40 mg) were added to a solution of 2 (742 mg, 2.98 mmol) in
MeCN (25 mL), and the resulting mixture was stirred at 60°C overnight. The mixture was
neutralised with Et3N, concentrated and purified by silica gel chromatography (toluene–
EtOAc 1:1) to yield 2-azidoethyl 4,6-O-benzylidene-a-D-mannopyranoside (3, 707 mg)
as a mixture containing some unreacted starting material and also dibenzylidenated
material, which was used without further purification in the next step; NMR data: 13C, d
50.5, 63.5, 66.7, 68.4, 68.7, 70.8, 78.7, 100.5, 102.3, 126.3–137.1. A solution of this
mixture and dibutyltin oxide (626 mg, 2.52 mmol) in MeOH (50 mL) was refluxed for

Synthesis of Cryptococcus neoformans. IV
571
1 h, then concentrated and dried in vacuum. The residue was dissolved in DMF (50
mL) and p-methoxybenzyl chloride (341 mL, 2.52 mmol) and CsF (414 mg, 2.72 mmol)
were added, and the mixture was stirred at rt overnight. The mixture was diluted with
toluene, washed twice with a sat aq KF solution, dried and concentrated. Silica gel
chromatography (toluene–EtOAc 3:1) yielded 2-azidoethyl 4,6-O-benzylidene-3-O-p-
methoxybenzyl-a-D-mannopyranoside (4, 612 mg, 45% from 2) as a syrup; NMR data:
13C, d 50.7, 55.5, 63.9, 67.0, 69.0, 70.0, 73.1, 75.4, 78.8, 100.4, 101.9, 114.1–159.7.
The position of the p-methoxybenzyl group is proven by the downfield shift of H-2
after acetylation of 4 [d 5.4 (dd, 1H, H-2)]. A solution of 4 (227 mg, 0.50 mmol) in
70% aq AcOH (10 mL) was heated at 70°C for 3 h, then concentrated and co-con-
centrated twice with toluene. Purification by silica gel chromatography (toluene–
EtOAc 1:3) gave 2-azidoethyl 3-O-p-methoxybenzyl-a-D-mannopyranoside (5, 155 mg,
85%); NMR data: 13C, d 50.3, 55.1, 61.0, 64.7, 66.5, 67.7, 71.6, 72.7, 78.9, 99.7,
113.7–159.2. A 60% oil dispersion of sodium hydride (75 mg, 1.89 mmol) was washed
once with dry light petroleum. DMF (3 mL) was added, and thereafter a solution of 5
(155 mg, 0.42 mmol) in DMF (4 mL) was added dropwise at 0°C. After 15 min, benzyl
bromide (210 mL, 1.76 mmol) in DMF (3 mL) was added, and the solution was allowed
to reach rt. MeOH (0.5 mL) was carefully added and the mixture was diluted with
toluene, washed three times with H2O, dried and concentrated. Purification on a silica
gel column (toluene–EtOAc 9:1) gave 2-azidoethyl 2,4,6-tri-O-benzyl-3-O-p-methoxy-
benzyl-a-D-mannopyranoside (6, 235 mg, 88%) as a syrup; NMR data: 13C, d 50.4,
55.2, 66.3, 69.2, 71.8, 72.0, 72.7, 73.2, 74.6 (2C), 74.9, 79.6, 98.1, 113.6–158.9. DDQ
(173 mg, 0.76 mmol) was added at 0°C to a solution of 6 (374 mg, 0.58 mmol) in
CH2Cl2:H2O (19:1, 20 mL). After stirring for 3 h, the mixture was washed twice with
aq NaHCO3 and water. Concentration of the organic phase followed by silica gel
chromatography (toluene–EtOAc 6:1) afforded 7 (248 mg, 82%); [a]D + 22° (c 1.0,
CHCl3); NMR data: 13C, d 50.4, 66.3, 69.0, 71.1, 71.5, 72.9, 73.3, 74.6, 76.3, 78.1,
97.1, 127.3–138.1.
Anal. Calcd for C29H33N3O6: C, 67.04; H, 6.40. Found: C, 66.85; H, 6.23.
Ethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 ! 2)-6-O-acetyl-3-O-allyl-4-O-
benzyl-1-thio- -D-mannopyranoside (15) and ethyl (2,3,4-tri-O-benzyl- -D-xylopyr-
anosyl)-(1 !2)-3-O-allyl-6-O-benzyl-1-thio- -D-mannopyranoside (14b). A solu-
tion of 8[16] (3.00 g, 9.60 mmol) and dibutyltin oxide (2.87 g, 11.53 mmol) in MeOH
(200 mL) was refluxed for 1 h, then concentrated and dried in vacuum. The residue
was dissolved in DMF (200 mL). Allyl bromide (979 mL, 11.57 mmol) and CsF (1.89
g, 12.44 mmol) were added, and the mixture was stirred at rt overnight. The reaction
mixture was diluted with toluene and washed twice with a sat aq KF solution, dried and
concentrated. Silica gel chromatography (toluene–EtOAc 3:1) yielded ethyl 3-O-allyl-
4,6-O-benzylidene-1-thio-a-D-mannopyranoside (9, 2.92 g, 86%); NMR data: 13C, d
14.9, 25.0, 63.8, 68.7, 71.5, 71.9, 75.4, 79.1, 84.2, 101.6, 117.6, 126.1–128.9, 134.4,
137.5. Silver triflate (5.47 g, 21.28 mmol) dissolved in dry toluene was added at À40°C
to a stirred solution of 9 (3.00 g, 8.51 mmol), 10[17] (11.18 g, 21.28 mmol) and 2,6-di-
tert-butylpyridine (3.80 mL, 17.02 mmol) in distilled CH2Cl2 (250 ml) containing
˚
crushed molecular sieves (4 A). After 1 h, Et3N (2 mL) was added, and stirring was
continued for 15 min. The mixture was diluted with CH2Cl2, filtered through a pad of

572
Alpe, Oscarson, and Svahnberg
Celite, concentrated, and purified by silica gel chromatography (toluene–EtOAc 12:1)
to give crude ethyl (2,3,4-tri-O-benzoyl-b-D-xylopyranosyl)-(1 ! 2)-3-O-allyl-4,6-O-
benzylidene-1-thio-a-D-mannopyranoside (11). The crude disaccharide was dissolved in
MeOH (250 mL), 1 M methanolic NaOMe (10 mL) was added, and the mixture was
stirred for 4 h at rt. Dowex 50 (H+) ion-exchange resin was added, and the stirring was
continued for 30 min. Filtration and concentration of the mixture followed by silica gel
chromatography (toluene–EtOAc 1:3) gave ethyl (b-D-xylopyranosyl)-(1 ! 2)-3-O-
allyl-4,6-O-benzylidene-1-thio-a-D-mannopyranoside (12, 3.37 g, 82% over two steps)
as a white foam; NMR data: 13C, d 15.2, 25.9, 64.6, 65.5, 68.7, 69.7, 70.4, 73.2, 74.1,
1
74.8, 74.9, 79.8, 84.7, 101.1, 101.8, 118.9, 126.3–133.8, 134.1, 137.7; H, d 4.58 (d, 1
H, J1,2 = 9 Hz, H-1’), 5.32 (s, 1 H, H-1). A 60% oil dispersion of sodium hydride (1.28
g, 31.85 mmol) was washed once with dry light petroleum. DMF (15 mL) was added
followed by dropwise addition at 0°C of a solution of 12 (3.43 g, 7.08 mmol) in DMF
(60 mL). After 15 min, benzyl bromide (3.54 mL, 29.73 mmol) in DMF (60 mL) was
added and the solution was stirred for 1 h. MeOH (5 mL) was carefully added and the
mixture was diluted with toluene and washed three times with H2O, dried and con-
centrated. Purification on a silica gel column (toluene–EtOAc 15:1) gave ethyl (2,3,4-
tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-3-O-allyl-4,6-O-benzylidene-1-thio-a-D-man-
nopyranoside (13, 4.67 g, 87%) as a syrup; NMR data: 13C, d 15.0, 25.6, 64.0, 64.5,
68.7, 70.9, 73.3, 74.1, 75.1, 75.5, 77.7, 77.8, 78.9, 81.3, 83.2, 83.7, 101.5, 103.0, 117.0,
126.0–128.8, 134.7, 137.5–138.6. MALDI-TOF MS: m/z calcd for C44H50NaO9S
([M + Na]+): 777.31. Found 776.72.
A solution of AlCl3 (707 mg, 5.30 mmol) in diethyl ether (50 mL) was added
dropwise during 30 min to a stirred mixture of 13 (1.00 g, 1.32 mmol), BH3–tri-
˚
methylamine complex (3.86 g, 52.98 mmol) and 4 A molecular sieves in
CH2Cl2:diethyl ether (5:1, 120 mL) at 0°C. After 1 h, the mixture was filtered through
a pad of Celite and 1M H2SO4 was added to the filtrate, which then was stirred for 30
min. The phases were separated and the organic layer was washed with aq NaHCO3
and H2O, dried and concentrated. A short silica gel column gave a mixture of 14a and
14b, which was acetylated at the primary hydroxyl group before further purification.
The crude disaccharide mixture and sym-collidine (286 mL, 2.15 mmol) were dissolved
in CH2Cl2 (30 mL) and the solution was cooled to À70°C. Acetyl chloride (92 mL, 1.28
mmol) was added and the reaction was stirred for 10 min at À70°C, then quenched
with MeOH, and allowed to attain rt. Concentration of the mixture, followed by silica
gel chromatography (toluene–EtOAc 15:1), afforded 15 (366 mg, 35% over two steps)
together with 390 mg of ethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-3-O-al-
lyl-6-O-benzyl-1-thio-a-D-mannopyranoside (14b). 15: [a]D + 37° (c 1.0, CHCl3);
NMR data: 13C, d 15.0, 20.5, 25.7, 63.3, 64.1, 69.9, 70.5, 73.4, 74.0, 75.1, 75.2, 75.6,
76.9, 77.3, 78.4, 81.2, 82.5, 83.9, 103.5, 118.1, 127.6–128.9, 134.6, 138.0–138.8,
170.7; MALDI-TOF MS: m/z calcd for C46H54NaO10S ([M + Na]+): 821.33. Found
820.91.
Anal. Calcd for C46H54O10S: C, 69.15; H, 6.81. Found: C, 68.91; H, 6.61.
Ethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 !2)-3-O-allyl-4,6-di-O-benzyl-
1-thio- -D-mannopyranoside (16). A 60% oil dispersion of sodium hydride (41 mg,
1.03 mmol) was washed once with dry light petroleum. DMF (2 mL) was added, and a
solution of 14b (390 mg, 0.52 mmol) in DMF (10 mL) was added dropwise at 0°C.

Synthesis of Cryptococcus neoformans. IV
573
After 15 min, benzyl bromide (110 mL, 0.93 mmol) in DMF (5 mL) was added and the
solution was stirred for 4 h. MeOH (1 mL) was carefully added and the mixture was
diluted with toluene and washed three times with H2O, dried and concentrated. Puri-
fication on a silica gel column (toluene–EtOAc 15:1) gave 16 (378 mg, 34% over two
steps) as a syrup; [a]D + 48° (c 1.0, CHCl3); NMR data: 13C, d 15.0, 25.5, 64.1, 69.4,
70.5, 71.9, 73.1, 73.4, 74.7, 74.9, 75.2, 75.5, 77.0, 77.3, 78.5, 81.0, 82.3, 84.0, 103.5,
117.7, 127.3–129.0, 134.8, 138.3–138.8; MALDI-TOF MS: m/z calcd for C51H58
NaO9S ([M + Na]+): 869.37. Found 868.92.
Anal. Calcd for C51H58O9S: C, 72.31; H, 6.90. Found: C, 72.09; H, 7.11.
Ethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 !4)-[(2,3,4-tri-O-benzyl- -D-
xylopyranosyl)-(1 ! 2)]-3-O-allyl-6-O-benzyl-1-thio- -D-mannopyranoside (20).
A
˚
solution of 9 (832 mg, 2.36 mmol), NaCNBH3 (1.56 g, 23.6 mmol) and 3 A molecular
sieves in distilled THF (100 mL) was stirred at rt under argon for 30 min. HCl in
diethyl ether was added until pH = 1. After 30 min, the reaction mixture was filtered
through a layer of Celite, concentrated and purified on silica gel (toluene–EtOAc, 4:1)
to give ethyl 3-O-allyl-6-O-benzyl-1-thio-a-D-mannopyranoside (17, 624 mg, 1.76
mmol, 75%); NMR data: 13C, d 15.2, 25.4, 68.6, 69.8, 70.5, 71.1, 71.3, 74.0, 79.6, 84.0,
118.6, 128.1–128.8, 134.5, 138.2. Silver triflate (362 mg, 1.41 mmol) dissolved in dry
toluene was added at À40°C to a stirred solution of 17 (100 mg, 0.28 mmol), 10 (741
mg, 1.41 mmol) and 2,6-di-tert-butylpyridine (253 mL, 1.13 mmol) in distilled CH2Cl2
˚
(50 ml) containing crushed molecular sieves (4 A). After 2h, Et3N (1 mL) was added
and stirring was continued for 15 min. The mixture was diluted with CH2Cl2, filtered
through a pad of Celite, concentrated, and purified by silica gel chromatography
(toluene–EtOAc 10:1) to give crude ethyl (2,3,4-tri-O-benzoyl-b-D-xylopyranosyl)-
(1 ! 4)-[(2,3,4-tri-O-benzoyl-b-D-xylopyranosyl)-(1 ! 2)]-3-O-allyl-6-O-benzyl-1-
thio-a-D-mannopyranoside (18). The crude trisaccharide was dissolved in MeOH
(100 mL). 5 mL of 1 M methanolic NaOMe was added and the mixture was stirred
for 2 h at rt. Dowex 50 (H+) ion-exchange resin was added, and stirring was con-
tinued for 30 min. Filtration and evaporation, followed by silica gel chromatography
(CH2Cl2:MeOH 3:1), then gave ethyl (b-D-xylopyranosyl)-(1 ! 4)-[(b-D-xylopyr-
anosyl)-(1 ! 2)]-3-O-allyl-6-O-benzyl-1-thio-a-D-mannopyranoside (19, 113 mg, 65%
over two steps); NMR data: 13C, d 15.0, 25.8, 65.6, 65.7, 68.7, 69.5, 69.8, 71.4, 71.5,
72.1, 73.6, 73.7, 74.1, 75.3, 75.6, 76.4, 76.9, 82.7 (C-1), 102.2, 103.4 (C-1’, 1@),
118.3, 128.1–133.1, 134.4, 137.6; MALDI-TOF MS: m/z calcd for C29H42NaO13S
([M + Na]+): 641.22. Found 640.91.
A solution of 19 (113 mg, 0.18 mmol) and BnBr (260 mL, 2.19 mmol) in dry DMF
(10 mL) was added dropwise to a cold (0°C) suspension of NaH (110 mg, 2.75 mmol)
in DMF (10 mL). The mixture was stirred at rt for 4 h before the addition of MeOH (2
mL). Toluene was added, and the mixture was washed with sat aq NaHCO3 and water.
The organic layer was dried, concentrated and purified by silica gel chromatography
(toluene–EtOAc, 10:1) to give 20 (129 mg, 0.11 mmol, 61%); [a]D + 52° (c 1.0,
CHCl3); NMR data: 13C, d 15.0, 25.5, 63.7, 64.0, 68.9, 71.3, 71.9, 72.9, 73.1, 73.3,
74.7, 74.9, 75.5, 76.5, 76.7, 77.0, 77.5, 78.2, 81.1, 82.0, 82.3, 84.0, 84.2, 103.1, 103.3,
116.8, 127.3–129.0, 135.3, 138.2–138.9; MALDI-TOF MS: m/z calcd for C70H78
NaO13S ([M + Na]+): 1181.51. Found 1180.95.
Anal. Calcd for C70H78O13S: C, 72.51; H, 6.78. Found: C, 72.30; H, 6.91.

574
Alpe, Oscarson, and Svahnberg
2-Azidoethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 !2)-(4,6-di-O-benzyl- -
D-mannopyranosyl)-(1 ! 3)-[(2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 ! 2)]-(6-O-
acetyl-4-O-benzyl- -D-mannopyranosyl)-(1 !3)-2,4,6-tri-O-benzyl- -D-mannopyr-
anoside (24). A solution of 7 (102 mg, 0.20 mmol) and 15 (235 mg, 0.29 mmol) in
˚
dry diethyl ether (10 mL) containing powdered molecular sieves (4 A) was stirred at rt
in an argon atmosphere for 30 min. DMTST (203 mg, 0.78 mmol) was added to the
mixture, and stirring was continued for 6 h. After neutralization with Et3N, the mixture
was filtered through a pad of Celite and concentrated. The residue was purified on a
silica gel column (toluene–EtOAc 6:1) to yield crude 2-azidoethyl (2,3,4-tri-O-benzyl-
b-D-xylopyranosyl)-(1 ! 2)-(6-O-acetyl-3-O-allyl-4-O-benzyl-a-D-mannopyranosyl)-
(1 ! 3)-(2,4,6-tri-O-benzyl-a-D-mannopyranoside) (21, 163 mg, 65%). PdCl2 (60%, 3
mg, 9.8 mmol) was added to a solution of 21 (123 mg, 0.098 mmol) in EtOH:MeOH
(1:1, 6 mL). The mixture was stirred for 7 h, then filtered through a pad of Celite,
concentrated and purified on a silica gel column (toluene–EtOAc 5:1) to yield 2-
azidoethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-(6-O-acetyl-4-O-benzyl-a-D-
mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (22, 71 mg, 60%);
NMR data: 13C, d 20.8, 50.6, 63.7, 64.1, 66.8, 69.2, 70.0, 71.1, 72.6, 72.8, 73.6, 73.7,
74.6, 74.9, 75.0, 75.2, 75.8, 76.4, 77.5, 77.5, 78.9, 80.4, 81.3, 83.3, 98.1 (C-1), 100.5
1
(C-1’), 104.0 (C-1@), 126.9–138.9, 170.9; H, d 5.15 (H-1’). A solution of 22 (65 mg,
0.053 mmol) and 16 (68 mg, 0.080 mmol) in dry diethyl ether (5 mL) containing
˚
powdered molecular sieves (4 A) was stirred at rt in an argon atmosphere for 30 min.
DMTST (55 mg, 0.21 mmol) was added to the mixture, and the stirring was continued
for 5 h. After neutralization with Et3N, the mixture was filtered through a pad of Celite
and concentrated. The residue was purified on a silica gel column (toluene–EtOAc 9:1)
to yield 2-azidoethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-(3-O-allyl-4,6-di-
O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-[(2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-
(1 ! 2)]-(6-O-acetyl-4-O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-
D-mannopyranoside [23, 69 mg, 65% (95% based on consumed acceptor)] together with
21 mg of unreacted 22; NMR data 23: 13C, d 20.3, 50.2, 62.9–83.1, 97.4 (C-1), 99.8,
100.3 (C-1’, 1000), 103.4 (2C, C-1@, 10000), 116.4, 126.4–128.5, 135.0, 137.6–139.1,
170.4. PdCl2 (60%, 1 mg, 3.2 mmol) was added to a solution of 23 (60 mg, 0.030
mmol) in EtOH:MeOH (1:1, 6 mL). The mixture was stirred for 5 h, filtered through a
pad of Celite, concentrated and purified on a silica gel column (toluene–EtOAc 6:1) to
yield 24 (47 mg, 80%); [a]D + 10° (c 1.0, CHCl3); NMR data: 13C, 20.5, 50.4, 63.1–
83.2, 97.6 (C-1), 100.3, 100.7 (C-1’, 1000), 103.6, 103.9 (C-1@,10000), 126.6–139.2, 170.6;
1H, d 5.14, 5.28 (H-1’, 1000); MALDI-TOF MS: m/z calcd for C116H125N3NaO25
([M + Na]+): 1982.85. Found 1983.05.
Anal. Calcd for C116H125N3O25: C, 71.04; H, 6.42. Found: C, 70.84; H, 6.62.
2-Azidoethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 !2)-(4,6-di-O-benzyl- -
D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl- -D-mannopyranoside (26). A solu-
tion of 7 (44 mg, 0.085 mmol) and 16 (100 mg, 0.12 mmol) in dry diethyl ether (5 mL)
˚
containing powdered molecular sieves (4 A) was stirred at rt in an argon atmosphere for 30
min. DMTST (87 mg, 0.34 mmol) was added to the mixture, and stirring was continued for 3
h. After neutralization with Et3N, the mixture was filtered through a pad of Celite and
concentrated. The residue was purified on a silica gel column (toluene–EtOAc 12:1) to
yield 2-azidoethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-(3-O-allyl-4,6-di-O-
benzyl-a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (25, 100

Synthesis of Cryptococcus neoformans. IV
575
mg, 91%); NMR data: 13C, d 50.4, 63.9–83.5, 97.8 (C-1), 99.9 (C-1’), 103.6 (C-1@), 117.2,
126.8–129.1, 135.1, 138.2–139.0. PdCl2 (60%, 1 mg, 3.2 mmol) was added to a solution
of 25 (41 mg, 0.031 mmol) in EtOH:MeOH (1:1, 6 mL). The mixture was stirred for
5 h, filtered through a pad of Celite, concentrated and purified on a silica gel column
(toluene–EtOAc 9:1) to yield 26 (29 mg, 73%); [a]D + 33° (c 1.0, CHCl3); NMR
data: 13C, d 50.4, 63.9, 66.6, 69.0, 69.4, 70.7, 72.2, 72.4, 72.5, 73.3, 73.3, 73.4, 74.4,
74.6, 74.7, 74.9, 75.5, 77.2, 77.5, 80.4, 80.7, 83.2, 97.9 (C-1), 100.5 (C-1’), 103.9 (C-
1
1@), 126.8–128.7, 138.1–138.9; H, d 5.22 (H-1’); MALDI-TOF MS: m/z calcd for
C75H81N3NaO15 ([M + Na]+): 1286.56. Found 1286.17.
Anal. Calcd for C75H81N3O15: C, 71.24; H, 6.46. Found: C, 71.03; H, 6.67.
2-Azidoethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 !2)-(4,6-di-O-benzyl- -
D-mannopyranosyl)-(1 ! 3)-[(2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 ! 2)]-(4,6-di-
O-benzyl- -D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl- -D-mannopyranoside
(28). A solution of 26 (76 mg, 0.060 mmol) and 16 (74 mg, 0.087 mmol) in dry
˚
diethyl ether (10 mL) containing powdered molecular sieves (4 A) was stirred at rt in
an argon atmosphere for 30 min. To the mixture was added DMTST (63 mg, 0.24
mmol) and the stirring was continued for 2 h. After neutralization with Et3N, the
mixture was filtered through a pad of Celite, and concentrated. The residue was
purified on a silica gel column (toluene–EtOAc 9:1) to yield crude 2-azidoethyl (2,3,4-
tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)-(3-O-allyl-4,6-di-O-benzyl-a-D-mannopyr-
anosyl)-(1 ! 3)-[(2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)]-(4,6-di-O-benzyl-
a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (27, 105 mg,
85%). PdCl2 (60%, 1 mg, 3.2 mmol) was added to a solution of 27 (105 mg, 0.051
mmol) in EtOH:MeOH (1:1, 6 mL). The mixture was stirred for 5 h, filtered through a
pad of Celite, concentrated and purified on a silica gel column (toluene–EtOAc 9:1) to
yield 28 (64 mg, 62%); [a]D + 9° (c 1.0, CHCl3); NMR data: 13C, d 50.6, 63.4–83.5,
97.9 (C-1), 100.7, 100.8 (C-1’, 1000), 103.9, 104.1 (C-1@, 10000), 126.4–128.7, 138.4–
1
139.5; H, d 5.18, 5.28 (H-1’, 1000); MALDI-TOF MS: m/z calcd for C121H129N3NaO24
([M + Na]+): 2030.89. Found 2030.73.
Anal. Calcd for C121H129N3O24; C, 72.33; H, 6.47. Found: C, 72.19; H, 6.62.
2-Azidoethyl (2,3,4-tri-O-benzyl- -D-xylopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-ben-
zyl- -D-xylopyranosyl)-(1 !2)]-(6-O-benzyl- -D-mannopyranosyl)-(1 !3)-[(2,3,4-
tri-O-benzyl- -D-xylopyranosyl)-(1 !2)]-(4,6-di-O-benzyl- -D-mannopyranosyl)-
(1 !3)-2,4,6-tri-O-benzyl- -D-mannopyranoside (30). A solution of 20 (33 mg, 29
mmol) and 26 (25 mg, 20 mmol) in dry diethyl ether (5 mL) containing powdered
˚
molecular sieves (4 A) was stirred at rt in an argon atmosphere for 30 min. To the
mixture was added DMTST (20 mg, 79 mmol), and stirring was continued for 30 min.
After neutralization with Et3N, the mixture was filtered through a pad of Celite and
concentrated. The residue was purified on a silica gel column (toluene–EtOAc 10:1) to
yield 2-azidoethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-
b-D-xylopyranosyl)-(1 ! 2)]-(3-O-allyl-6-O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-
[(2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 2)]-(4,6-di-O-benzyl-a-D-mannopyr-
anosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside side (29, 39 mg, 17 mmol,
85%); Selected NMR data: 13C, d 97.8 (C-1), 99.6, 100.2 (C-1’, 1000), 103.2, 103.3, 103.5
(C-1@, 10000, 100000); MALDI-TOF MS: m/z calcd for C143H153N3NaO28 ([M + Na]+):
2383.05, Found 2383.35. PdCl2 (60%, 5 mg, 17 mmol) was added to a solution of 29

576
Alpe, Oscarson, and Svahnberg
(18 mg, 7.6 mmol) in EtOH:MeOH (1:1, 4 mL). The mixture was stirred for 2 h,
filtered through a pad of Celite, concentrated and purified on a silica gel column
(toluene–EtOAc 10:1) to give 30 (11 mg, 62%); [a]D + 25° (c 1.0, CHCl3); Selected
NMR data: 13C, d 97.7 (C-1), 98.6, 100.2 (C-1’, 1000), 103.1, 103.4, 104.0 (C-1@, 10000,
1
100000); H, d 5.17, 5.29 (H-1’, 1000).
Anal. Calcd for C140H149N3O28: C, 72.43; H, 6.47. Found: C, 72.20; H, 6.61.
ACKNOWLEDGMENTS
Financial support from the Swedish Foundation for Strategic Research (the
‘‘Glycoconjugates in Biological Systems’’ program) and from the Swedish Natural
Science Research Council are gratefully acknowledged.
REFERENCES
1. Brummer, E. Human defences against Cryptococcus neoformans: an update.
Mycopathologia 1999, 143, 121–125.
2. Cherniak, R.; Sundstrom, B.J. Polysaccharide antigens of the capsule of
Cryptococcus neoformans. Infect. Immun. 1994, 62, 1507–1512.
3. Fromtling, R.A.; Shadomy, H.J.; Jacobson, E.S. Decreased virulence in stable
capsular mutants of Cryptococcus neoformans. Mycopathologia 1982, 79, 23–29.
4. Aberg, J.A.; Powderly, W.G. Cryptococcosis. Adv. Pharmacol. 1997, 37, 215–251.
5. Doering, T.L. How does Cryptococcus get its coat? Trends Microbiol. 2000, 8,
547–553.
6. Bhattacharjee, A.K.; Bennet, J.E.; Glaudemans, C.P.J. Capsular polysaccharides of
Cryptococcus neoformans. Rev. Infect. Dis. 1984, 6, 619–624.
7. Belay, T.; Cherniak, R. Determination of antigen binding specificities of
Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay.
Infect. Immun. 1995, 63, 1810–1819.
8. Olsson, L. Synthesis of oligosaccharide fragments from Cryptococcus neoformans
polysaccharide capsule and preparation of glucuronic acid thioglycosides for b-
glucuronide synthesis. Licentiate thesis; Stockholm University, 1994.
9. Olsson, L. Synthesis of oligosaccharides related to the capsular polysaccharide of
Cryptococcus neoformans. Doctoral thesis; Stockholm University, 1996.
10. Garegg, P.J.; Olsson, L.; Oscarson, S. Synthesis of oligosaccharides corresponding
to structures found in capsular polysaccharides of Cryptococcus neoformans. J.
Carbohydr. Chem. 1993, 12, 955–967.
11. Garegg, P.J.; Olsson, L.; Oscarson, S. Synthesis of oligosaccharides corresponding
to structures found in capsular polysaccharides of Cryptococcus neoformans. Part 2.
Bioorg. Med. Chem. 1996, 4, 1867–1871.
12. Garegg, P.J.; Olsson, L.; Oscarson, S. Synthesis of oligosaccharides corresponding
to structures found in capsular polysaccharides of Cryptococcus neoformans. Part 3.
A regioselectively acetylated tetrasaccharide. J. Carbohydr. Chem. 1997, 16, 973–
981.

Synthesis of Cryptococcus neoformans. IV
577
13. Zhang, J.; Kong, F. An efficient synthesis of a hexasaccharide—the repeating unit
of the exopolysaccharide from Cryptococcus neoformans serovar A. Tetrahedron
Lett. 2003, 44, 1839–1842.
14. Chernyak, A.Y.; Sharma, G.V.M.; Kononov, L.O.; Krishna, P.R.; Levinsky, A.B.;
Kochetkov, N.K.; Rama Rao, A.V. 2-Azidoethyl glycosides: glycosides potentially
useful for the preparation of neoglycoconjugates. Carbohydr. Res. 1992, 223, 303–
309.
15. Zegelaar-Jaarsveld, K.; Smits, S.A.W.; van der Marel, G.A.; van Boom, J.H.
Synthesis of a pentasaccharide corresponding to the repeating unit of the exo-
polysaccharide from Cryptococcus neoformans serovar D. Bioorg. Med. Chem.
1996, 4, 1819–1832.
16. Garegg, P.J.; Kvarnstro¨m, I.; Niklasson, A.; Niklasson, G.; Svensson, S.C.T. Partial
substitution of thioglycosides by phase-transfer catalysed benzoylation and
benzylation. J. Carbohydr. Chem. 1993, 12, 933–953.
17. Fletcher, H.G.; Hudson, C.S. 1,5-Anhydro-xylitol. J. Am. Chem. Soc. 1947, 69,
921–924.
18. Andrews, C.W.; Rodebaugh, R.; Fraser-Reid, B. A solvation-assisted model for
estimating anomeric reactivity. Predicted versus observed trends in hydrolysis of n-
pentenyl glycosides. J. Org. Chem. 1996, 61, 5280–5289.
19. Ek, M.; Garegg, P.J.; Hultberg, H.; Oscarson, S. Reductive ring openings of
carbohydrate benzylidene acetals using borane-trimethylamine and aluminium
chloride. Regioselectivity and solvent dependence. J. Carbohydr. Chem. 1983, 2,
305–311.
20. Ishihara, K.; Kurihara, H.; Yamamoto, H. An extremely simple, convenient, and
selective method for acetylating primary alcohols in the presence of secondary
alcohols. J. Org. Chem. 1993, 58, 3791–3793.
21. Lindhorst, T.; Ko¨tter, S.; Krallmann-Wenzel, U.; Ehlers, S. Trivalent a-D-man-
noside clusters as inhibitors of type-1 fimbriae-mediated adhesion of Escherichia
coli: structural variation and biotinylation. J. Chem. Soc., Perkin Trans. 1 2001,
823–831.
Received February 25, 2003
Accepted July 23, 2003