Synthesis of Cryptococcus neoformans capsular polysaccharide structures. Part V: Construction of glucuronic acid-containing thioglycoside donor blocks

Article abstract of DOI:10.1081/CAR-200040114

Glucuronic acid-containing di- and trisaccharide thioglycoside building blocks, ethyl (benzyl 2,3,4-tri-O-benzyl-β-D-glucopyranosyluronate)-(1 → 2)-3-O-allyl-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside, ethyl (benzyl 2,3,4-tri-O-benzyl-β-D-glucopyranosyluronate)-(1 → 2)-6-O-acetyl-3-O-allyl-4-O-benzyl-1-thio-α-D-mannopyranoside and ethyl (2,3,4-tri-O-benzyl-β-D-xylopyranosyl)-(1 → 4)-[(benzyl 2,3,4-tri-O-benzyl-β-D-glucopyranosyluronate)-(1 → 2)]-3-O-allyl-6-O-benzyl-1-thio-α-D-mannopyranoside, corresponding to repetitive structures in the capsular polysaccharide (CPS) of Cryptococcus neoformans, have been synthesized. The blocks contain an orthogonal allyl group in the 3-position of the mannose residue to allow formation of the (1 → 3)-linked mannan backbone of the CPS and benzyl ethers as persistent protecting groups to facilitate access to acetylated target structures. The glucuronic acid moiety was introduced using an acetylated trichloroacetimidate donor and the xylose residue employing the benzoylated bromo sugar to ensure β-selectivity in the couplings. Exchange to benzyl protecting groups was then performed at the di- or trisaccharide level. Assembly of suitable blocks employing DMTST as promoter in diethyl ether then afforded, in high yield and with stereoselectivity, a protected pentasaccharide corresponding to a C. neoformans serotype D CPS structure. Copyright

Full text of DOI:10.1081/CAR-200040114

This article was downloaded by: [Linnaeus University]
On: 07 October 2014, At: 11:08
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Carbohydrate Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lcar20
Synthesis of Cryptococcus neoformans
Capsular Polysaccharide Structures.
Part V: Construction of Glucuronic
Acid‐Containing Thioglycoside Donor
Blocks
Mia Alpe a , Stefan Oscarson a & Pär Svahnberg a
a Department of Organic Chemistry, Arrhenius Laboratory Floor 6 ,
Stockholm University , S-106 91, Stockholm, Sweden
Published online: 18 Aug 2006.
To cite this article: Mia Alpe , Stefan Oscarson & Pär Svahnberg (2004) Synthesis of Cryptococcus
neoformans Capsular Polysaccharide Structures. Part V: Construction of Glucuronic Acid‐Containing
Thioglycoside Donor Blocks, Journal of Carbohydrate Chemistry, 23:6-7, 403-416, DOI: 10.1081/
CAR-200040114
To link to this article: http://dx.doi.org/10.1081/CAR-200040114
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 6&7, pp. 403–416, 2004
Synthesis of Cryptococcus neoformans
Capsular Polysaccharide Structures. Part V:
Construction of Glucuronic Acid-Containing
Thioglycoside Donor Blocks
*
Mia Alpe, Stefan Oscarson, and Par Svahnberg
¨
Department of Organic Chemistry, Arrhenius Laboratory Floor 6, Stockholm
University, Stockholm, Sweden
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 405
III. EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
*
Correspondence: Stefan Oscarson, Department of Organic Chemistry, Arrhenius Laboratory Floor
6, Stockholm University, S-106 91 Stockholm, Sweden; Fax: þ46 8 15 49 08; E-mail: s.ocarson@
organ.su.se.
403
DOI: 10.1081/CAR-200040114
0732-8303 (Print); 1532-2327 (Online)
www.dekker.comf
Copyright # 2004 by Marcel Dekker, Inc.

404
Alpe, Oscarson, and Svahnberg
ABSTRACT
Glucuronic acid-containing di- and trisaccharide thioglycoside building blocks, ethyl
(benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-4,6-di-O-
benzyl-1-thio-a-D-mannopyranoside, ethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyra-
nosyluronate)-(1 ! 2)-6-O-acetyl-3-O-allyl-4-O-benzyl-1-thio-a-D-mannopyranoside
and ethyl (2,3,4-tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 4)-[(benzyl 2,3,4-tri-O-benzyl-
b-D-glucopyranosyluronate)-(1 ! 2)]-3-O-allyl-6-O-benzyl-1-thio-a-D-mannopyrano-
side, corresponding to repetitive structures in the capsular polysaccharide (CPS) of
Cryptococcus neoformans, have been synthesized. The blocks contain an orthogonal
allyl group in the 3-position of the mannose residue to allow formation of the
(1 ! 3)-linked mannan backbone of the CPS and benzyl ethers as persistent protecting
groups to facilitate access to acetylated target structures. The glucuronic acid moiety
was introduced using an acetylated trichloroacetimidate donor and the xylose residue
employing the benzoylated bromo sugar to ensure b-selectivity in the couplings.
Exchange to benzyl protecting groups was then performed at the di- or trisaccharide
level. Assembly of suitable blocks employing DMTST as promoter in diethyl ether
then afforded, in high yield and with stereoselectivity, a protected pentasaccharide
corresponding to a C. neoformans serotype D CPS structure.
Key Words: Oligosaccharide synthesis; Convergent synthesis; Glycoconjugate
vaccines.
INTRODUCTION
Cryptococcus neoformans is an opportunistic species causing severe diseases and
death, especially in immunodeficient patients.[1,2] The fungi is surrounded by a thick
layer of capsular polysaccharides, which is important for its virulence.[3–5] The major
CPS is built up by an a-(1 ! 3)-mannan backbone substituted with b-xylose residues
in the 2- and 4-positions and b-glucuronic acid residues in the 2-positions (Fig. 1).[6,7]
C. neoformans is divided into four serotypes, A–D, and the different serotypes are
defined by the amount and position of the xylose residues. The CPS also contains
acetyl groups at the 6-positions of the mannan backbone, but, although the acetylation
Figure 1. Schematic structure of deacetylated Cryptococcus neoformans CPS.

Synthesis of Cryptococcus neoformans Structures
405
pattern is believed to be immunologically important, little is known about the distribution
of the acetyl groups and its influence on the immune response.[7]
To investigate in more detail the immunological determinants of the C. neoformans
CPS and to construct glycoconjugate vaccine candidates, the development of an efficient
route to well-defined synthetic oligosaccharide structures corresponding to fragments of
the native CPS would be of great value.
Due to the repetitive and similar structure of the various C. neoformans CPS, a block
synthesis approach looks most attractive. With only six building blocks, four disaccharides
(I, II, IV, and V) and two trisaccharides (III and VI) (Fig. 2), it should be possible to con-
struct any structure wanted. In an earlier publication, we described the syntheses of three
xylose-containing thioglycoside building blocks (I–III) and their efficient assembly to
form penta- and hexasaccharide part structures from serotype A, B, and C CPSs.[8]
Herein, we report the construction of three glucuronic acid-containing blocks (IV–VI)
and an example of the use of block IV as a donor and subsequent acceptor to afford a
fully protected pentasaccharide as a representative intermediate to deacetylated targets
of serotype D structures.
RESULTS AND DISCUSSION
A strategy similar to that of the earlier synthesized blocks was applied, that is, the
use of thioglycoside donors with benzyl ethers as persistent protecting groups and allyl
ethers as temporary protecting groups. However, the formation of the uronic acid-
containing building blocks turned out to be quite complicated, in large part due to the
(possible) presence of acetate groups in the target compounds. Not only does this
more or less disqualify ester-protecting groups, but it also means that the carboxyl
moiety must be protected as an orthogonal ester. Furthermore, it is known that benzyla-
tion of uronic acid derivatives is troublesome.[9] Taking all this into consideration, we
decided to start with a perbenzylated uronic acid derivative as donor, in spite of the
fact that such precursor may not be that easily available and of the obvious problems
associated with the necessity for b-selectivity in the coupling reactions. The mannose
derivative 1, earlier used in the formation of the xylose-containing building blocks,[8]
was chosen as acceptor. However, all attempts to construct the desired b-linked disac-
charide in an acceptable yield failed. The thioglycoside donor at best (promotor NIS/
TfOH) gave a 3 : 1 a/b-ratio in 80% yield, whereas the trichloroacetimidate donor
gave exclusively the a-linked disaccharide.[10] Because of this, the use of a glucose
donor and introduction of the carboxyl group at the disaccharide level was investigated,
but severe problems were encountered in the oxidation step, partly due to concomitant
oxidation of the thioglycoside.[11]
Therefore, a third approach was tried using glucuronic acid donors with participating
protecting groups. The trichloroacetimidate donor 2,[12] easily available from glucuronic
acid 3,6-lactone in four efficient steps, was coupled to acceptor 1[8] to afford the b-linked
disaccharide 3 in 58% yield (Sch. 1). Our earlier attempts to benzylate (NaH, BnBr, DMF)
glucuronic acid derivatives have resulted in no product at all, even when starting from the
carboxylic acid salt.[9] However, a change of solvent to DMSO, as described by Koto
et al.,[13] has proven to give reasonable yields of products. Removal of the acetates in
compound 3 with sodium methoxide gave the methyl ester triol, which was benzylated

406
Alpe, Oscarson, and Svahnberg

Synthesis of Cryptococcus neoformans Structures
407

408
Alpe, Oscarson, and Svahnberg
(NaH, BnBr, DMSO). During this reaction only partial transesterification was observed
giving a mixture of the benzyl ester 4 and the corresponding methyl ester as product.
Hence, the methyl ester was saponified prior to benzylation, which afforded the pure
benzyl ester 4 (45% overall yield from 3). Reductive opening of the benzylidene acetal
in compound 4, using BH3-Me3N/AlCl3,[14] gave an inseparable 1 : 1 mixture of the
4-OH and 6-OH derivatives. Regioselective acetylation[19] of the obtained mixture
afforded an easily separable mixture of compound 5 (30%) and the first building block
6 (compare IV in Fig. 2) (32%).
From compound 5, the two other building blocks V and VI are obtainable through
benzylation or introduction of a xylose residue at the 4-position, respectively. However,
both these blocks were more efficiently synthesized starting from derivative 3 (Sch. 2).
Regioselective reductive opening (NaCNBH3/HCl) of the benzylidene group in 3 gave
the 6-O-benzyl derivative 7 (71%). Methoxide treatment of 7 afforded the methyl ester
tetraol. This time no saponification step was needed because it was found that the
methyl ester was completely hydrolyzed during purification by silica gel chromatography.
Subsequent benzylation gave the second building block 8 (compare V in Fig. 2) in 50%
overall yield.
The third building block (VI) was also obtained from 7 by a silver triflate-promoted
coupling with benzobromoxylose[15] (9) to obtain trisaccharide 10 (47%). In spite of a
two-fold (or larger) excess of donor used, 35% of the acceptor was recovered after the
coupling reaction. In the following deacylation step, the methyl ester was once more
lost during silica gel chromatography. Subsequent benzylation afforded the trisaccharide
building block 11 (24% from 10) together with building block 8 (17%), due to an unex-
pected loss of the xylose moiety under the benzylation conditions.
In spite of the rather low yields in the benzylation steps, this approach is so far the
shortest and most convenient one as long as there are no methods available to introduce
the perbenzylated glucuronic acid residue with good b-selectivity. Intermediate 3 is
easily obtained on a large scale, and the three different building blocks may be prepared
in only three or four synthetic steps from this precursor.
Having all the three desired glucuronic acid-containing building blocks in hand, their
donor and (following deallylation) acceptor qualities could next be tested. Our assumption
was that the 4,6-O-benzylidene acetal in the mannose confers low donor reactivity,[16]
which could explain some earlier failures.[17] To evaluate this hypothesis the couplings
of donors 4 and 8 to acceptor 12[8] were compared using dimethyl(methylthio)sulfonium
triflate (DMTST) as promoter. As expected, donor 8 reacted smoothly to give the trisac-
charide 13 in 81% yield (Sch. 3), whereas donor 4 did not react at all. However, using the
more efficient promoter system NIS/AgOTf, 4 could be activated to give a trisaccharide in
good yield (58% including deallylation).[10]
Deallylation of 13, using conditions (PdCl2) worked out for the xylose-containing
oligosaccharides,[8] gave compound 14 (74%), which was shown to be an excellent accep-
tor in a coupling with donor 15[8] to give the protected repeating unit of C. neoformans
type D CPS (16) in 88% yield. This structure has earlier been synthesized by others,[18]
but their pathway does not include the possibility of elongation or the presence of
acetyl groups or conjugation of the unprotected target structures. However, the agreement
between 13C-data is excellent, proving the structure of compound 16.
In conclusion, we now have access to the necessary building blocks for the con-
struction of all possible structures from C. neoformans serotypes A–D. The efficacy

Synthesis of Cryptococcus neoformans Structures
409

410
Alpe, Oscarson, and Svahnberg

Synthesis of Cryptococcus neoformans Structures
411
as donors and their efficient transformation into and subsequent use as acceptors was
shown in the assembly of the protected repeating unit of deacetylated type D CPS.
The latter was obtained through two subsequent stereoselective and high-yielding coup-
lings intercepted by a deallylation step to yield the target pentasaccharide in an overall
53% yield from the available building blocks. The quest for all the repeating units
(with different end groups) of the various serotypes as well as larger structures can
now be launched to give an array of well-defined oligosaccharides to be used in
biological experiments to try to understand the immunology of the Cryptococcus
neoformans capsule.
EXPERIMENTAL
General methods. TLC was carried out on Merck precoated 60 F254 plates using UV-
light and/or 8% 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 ¼ 0.00) at 258C on a Varian 300 MHz or 400 MHz instrument. MALDI-TOF
spectra were recorded on a Bruker Biflex III instrument using 20,40,60-trihydroxyacetophe-
none trihydrate (THAP) as matrix. Organic phases were dried over Na2SO4 before evap-
oration, which was performed under reduced pressure.
Ethyl (methyl 2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-
4,6-O-benzylidene-1-thio-a-D-mannopyranoside (3). A solution of 2[12] (623 mg,
1.30 mmol) and 1[8] (353 mg, 1.00 mmol) in dry CH2Cl2 containing 4 A molecular
˚
sieves (0.6 g) was stirred at rt for 30 min and then cooled to –208C. Trimethylsilyl trifluor-
omethanesulfonate (0.45 mL of a 1 M solution in CH2Cl2) was added. After 2 h, the solu-
tion was neutralized by addition of N,N-diisopropyl-N-ethylamine, filtered through Celite,
and concentrated. Purification by silica gel chromatography (toluene-EtOAc 5 : 1) gave 3
(387 mg, 58%); [a]D þ26 (c 1.0, CHCl3); NMR data: 13C, d 15.2 (CH3CH2S), 20.8, 20.9,
21.0 (CH3CO ꢀ 3), 26.0 (CH3CH2S), 53.1 (CH3O), 64.9, 68.7, 69.6, 70.8, 71.4, 71.9, 72.9,
74.4, 78.3, 78.6 (C-2-6, C-20-50, CH2CHCH2O), 83.2 (C-1), 99.7, 102.0 (C-10, PhCH),
117.6 (CH2CHCH2O), 126.4–129.3 (aromatic C), 134.9 (CH2CHCH2O), 137.7 (ipso-
Bn), 167.2, 169.4, 169.7, 170.5 (CH3CO ꢀ 3, COOCH3); 1H, d 4.62 (d, 1H,
J1,2 ¼ 8.0 Hz, H-10); MALDI-TOF MS: m/z calcd for C31H40NaO14S ([M þ Na]þ):
691.2. Found: 691.2.
Ethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-
4,6-O-benzylidene-1-thio-a-D-mannopyranoside (4). Compound 3 (200 mg, 0.30 mmol)
was dissolved in MeOH and the pH was adjusted to 12 by treatment with 1 M methanolic
NaOMe. The mixture was stirred at rt for 30 min when 1 mL H2O was added. After an
additional 30 min the reaction mixture was concentrated, co-evaporated twice with
toluene, and purified on a short silica gel column (CHCl3-MeOH 3 : 1). This residue
was then dissolved in dry DMSO (6 mL) containing benzyl bromide (356 mL,
3.0 mmol) and stirred at 158C for 10 min before NaH (60% dispersion in oil, 120 mg,
3.0 mmol) was added. The reaction was stirred for 40 min at rt and then quenched by
the addition of toluene (165 mL) and water (55 mL). The organic layer was separated,
washed with HOAc (5% aqueous) and brine, dried, and concentrated. Silica gel chromato-
graphy (toluene-EtOAc 9 : 1) gave 4 (120 mg, 0.14 mmol, 45%); [a]D þ22 (c 1.0, CHCl3);
NMR data: 13C, d 14.9 (CH3CH2S), 25.6 (CH3CH2S), 64.4, 67.2, 68.6, 70.2, 74.0, 74.7,

412
Alpe, Oscarson, and Svahnberg
75.0, 75.2, 75.7, 78.5, 78.8, 81.2, 83.0, 83.8 (C-1, C-2-6, C-20-50, CH2CHCH2O,
PhCH2 ꢀ 4), 101.6, 102.5 (C-10, PhCH), 117.1 (CH2CHCH2O), 125.2–138.3 (aromatic
C, CH2CHCH2O), 167.6 (COOBn); MALDI-TOF MS: m/z calcd for C52H56NaO11S
([M þ Na]þ): 911.3. Found: 911.0.
Anal. Calcd for C52H56O11S: C, 70.25; H, 6.35. Found: C, 70.37; H, 6.42.
Ethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-
6-O-benzyl-1-thio-a-D-mannopyranoside (5) and ethyl (benzyl 2,3,4-tri-O-benzyl-b-
D-glucopyranosyluronate)-(1 ! 2)-6-O-acetyl-3-O-allyl-4-O-benzyl-1-thio-a-D-man-
nopyranoside (6). A solution of AlCl3 (33 mg, 0.25 mmol) in diethyl ether (3 mL)
was added dropwise during 30 min to a stirred mixture of 4 (56 mg, 0.06 mmol),
˚
BH3-trimethylamine complex (177 mg, 2.5 mmol) and 4 A molecular sieves in
CH2Cl2 : diethyl ether (5 : 1, 7 mL) at 08C. After 1 h, the mixture was filtered through
Celite and 1 M H2SO4 was added to the filtrate, which then was stirred for 30 min.
The phases were separated and the organic layer was washed with NaHCO3 (aqueous
sat.) and water, dried, and concentrated. Silica gel chromatography (toluene-
EtOAc 3 : 1) gave a mixture of 5 and ethyl (benzyl (2,3,4-tri-O-benzyl-b-D-glucopyrano-
syluronate)-(1 ! 2)-3-O-allyl-4-O-benzyl-1-thio-a-D-mannopyranoside, which was
submitted to a selective acetylation of the primary hydroxyl before further purification.
The crude disaccharide mixture and sym-collidine (14 mL, 0.11 mmol) were dissolved
in CH2Cl2 (5 mL) and the solution was cooled to –708C. Acetyl chloride (8 mL,
0.11 mmol) was added and the reaction was stirred for 10 min at –708C, then quenched
with MeOH and allowed to reach rt. Concentration of the mixture followed by
silica gel chromatography (toluene EtOAc 9 : 1) afforded 6 (19 mg, 20 mmol, 32%
over two steps) together with 5 (15 mg, 17 mmol, 30%). 6: [a]D þ22 (c 1.0, CHCl3);
NMR data: 13C, d 15.0 (CH3CH2S), 20.5 (CH3CO), 25.8 (CH3CH2S), 63.3, 67.4,
69.8, 70.0, 73.9, 74.7, 75.1, 75.1, 75.2, 75.8, 76.4, 78.3, 78.7, 81.1, 82.3, 84.0 (C-1-6,
C-20-50, CH2CHCH2O, PhCH2 ꢀ 5), 102.8 (C-10), 118.0 (CH2CHCH2O), 127.6–129.1
(aromatic C), 134.5 (CH2CHCH2O), 137.8–138.4 (ipso-Bn), 167.8, 170.6 (CH3CO,
COOBn); MALDI-TOF MS: m/z calcd for C54H60NaO12S ([M þ Na]þ): 955.4. Found
955.4. 5: [a]D þ17 (c 1.0, CHCl3); NMR data: 13C, d 14.9 (CH3CH2S), 25.6
(CH3CH2S), 67.3, 67.4, 69.6, 70.2, 71.8, 73.4, 74.7, 75.0, 75.1, 75.5, 75.8, 78.8,
81.0, 82.1, 83.9 (C-1-6, C-20-50, CH2CHCH2O, PhCH2 ꢀ 5), 102.8 (C-10), 118.4
(CH2CHCH2O), 127.3–129.0 (aromatic C), 134.4 (CH2CHCH2O), 138.2–138.5
(ipso-Bn), 167.8 (COOBn); MALDI-TOF MS: m/z calcd for C52H58NaO11S
([M þ Na]þ): 913.4. Found: 913.4.
Anal. Calcd for C52H58O11S: C, 70.09; H, 6.56. Found: C, 69.85; H, 6.73.
Ethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-
4,6-di-O-benzyl-1-thio-a-D-mannopyranoside (8). A mixture of 3 (448mg, 0.67mmol),
˚
NaCNBH3 (421 mg, 6.7 mmol) and 3 A molecular sieves in distilled THF (30 mL) was
stirred at rt under an argon atmosphere for 30 min. A saturated solution of HCl in
diethyl ether was carefully added until the evolution of gas was no longer detected.
After 1 h, the reaction mixture was filtered through a layer of Celite, concentrated, and pur-
ified on a silica gel column (toluene-EtOAc 4 : 1) to give ethyl (methyl 2,3,4-tri-O-acetyl-
b-D-glucopyranosyluronate)-(1 ! 2)-3-O-allyl-6-O-benzyl-1-thio-a-D-mannopyranoside
(7, 321 mg, 0.48 mmol, 71%). NMR data: 13C, d 14.8 (CH3CH2S), 20.5, 20.5, 20.6
(CH3CO ꢀ 3), 25.4 (CH3CH2S), 52.7 (CH3O), 67.3, 69.2, 69.9, 70.3, 70.4, 71.5, 71.8,
72.5, 73.2, 75.2, 77.6, 81.4 (C-1-6, C-20-50, CH2CHCH2O, PhCH2), 99.0 (C-10), 118.0

Synthesis of Cryptococcus neoformans Structures
413
(CH2CHCH2O), 127.4–128.3 (aromatic C), 134.5 (CH2CHCH2O), 138.2 (ipso-Bn),
166.7, 169.1, 169.3, 170.2 (CH3CO ꢀ 3, COOCH3). MALDI-TOF MS: m/z calcd for
C31H42NaO14S ([M þ Na]þ): 693.2. Found 692.5. A solution of 7 (233 mg, 0.35 mmol)
in MeOH (5 mL) was treated with a catalytic amount of 1 M methanolic NaOMe at rt
for 30 min. Dowex 50 (Hþ) ion exchange resin was added and the mixture was then
filtered, concentrated, and purified on a silica gel column (CHCl3-MeOH 9 : 1). On
the column, the methyl ester was hydrolyzed to the corresponding acid, which was
directly benzylated. A mixture of this residue and benzyl bromide (356 mL, 3.0 mmol)
in dry DMSO (6 mL) was stirred at 158C. NaH (60% dispersion in oil, 120 mg,
3.0 mmol) was added. After 40 min more BnBr (356 mL, 3.0 mmol) and NaH
(120 mg, 3.0 mmol) were added and the reaction was stirred for 2 h at rt. The reaction
was quenched by the addition of toluene (165 mL) and water (55 mL). The organic
layer was separated, washed with HOAc (5% aqueous) and brine, dried, and concentrated.
Silica gel chromatography (toluene-EtOAc 9 : 1) gave 8 (170 mg, 0.17 mmol, 50% over
two steps); [a]D þ26 (c 1.0, CHCl3); NMR data: 13C, d 14.9 (CH3CH2S), 25.5
(CH3CH2S), 67.3, 69.4, 70.0, 71.7, 72.1, 73.1, 74.4, 74.7, 75.0, 75.2, 75.7, 76.5, 78.2,
78.7, 80.9, 82.0, 84.0 (C-1-6, C-20-50, CH2CHCH2O, PhCH2 ꢀ 6), 102.8 (C-10), 117.8
(CH2CHCH2O), 127.3–129.1 (aromatic C), 134.7 (CH2CHCH2O), 137.8–138.7
(ipso-Bn), 167.8 (COOBn); MALDI-TOF MS: m/z calcd for C59H64NaO11S
([M þ Na]þ): 1003.4. Found: 1002.5.
Anal. Calcd for C59H64O11S: C, 72.22; H, 6.57. Found: C, 72.00; H, 6.71.
Ethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-[(2,3,4-
tri-O-benzyl-b-D-xylopyranosyl)-(1 ! 4)]-3-O-allyl-6-O-benzyl-1-thio-a-D-manno-
pyranoside (11). Silver triflate (153 mg, 0.60 mmol) dissolved in dry toluene was added at
–408C to a stirred solution of 7 (160 mg, 0.24 mmol), 9[15] (313 mg, 0.60 mmol) and 2,6-
di-tert-butylpyridine (107 mL, 0.48 mmol) in distilled CH2Cl2 (15 mL) containing crushed
˚
molecular sieves (4 A). After 2 h, Et3N (1 mL) was added and the stirring was continued
for 15min. The mixture was diluted with CH2Cl2, filtered through Celite, concentrated, and
purified by silica gel chromatography (toluene-EtOAc 5 : 1) to give ethyl (methyl 2,3,4-
tri-O-acetyl-b-D-glucopyranosyluronate)-(1 ! 2)-[(2,3,4-tri-O-benzoyl-b-D-xylopyranosyl)-
(1 ! 4)]-3-O-allyl-6-O-benzyl-1-thio-a-D-mannopyranoside (10, 125 mg, 0.11 mmol,
47% (85% based on consumed acceptor)) together with unreacted 7 (72 mg). NMR
data: 13C, d 14.7 (CH3CH2S), 20.4, 20.4, 20.6, 25.2 (CH3CH2S), 52.7 (CH3O), 61.5
(C-50), 69.1, 69.2, 69.2, 70.5, 70.9, 70.9, 71.2, 71.9, 72.5, 72.7, 75.3, 76.2, 76.8, 81.1
(C-1-6, C-20-40, C-200-500, CH2CHCH2O, PhCH2), 99.3, 100.3 (C-10,100), 117.6
(CH2CHCH2O), 125.2–133.3 (aromatic C), 134.9 (CH2CHCH2O), 138.2 (ipso-Bn),
165.1, 165.2, 165.5, 166.7, 169.0, 169.2, 170.1 (PhCO ꢀ 3, CH3CO ꢀ 3, COOCH3).
MALDI-TOF MS: m/z calcd for C57H62NaO21S ([M þ Na]þ): 1137.3. Found 1136.5.
A solution of 10 (125 mg, 0.11 mmol) in MeOH (5 mL) was treated with a catalytic
amount of 1M methanolic NaOMe at rt for 30 min. Dowex 50 (Hþ) ion exchange
resin was added and the mixture was then filtered, concentrated, and purified on a
silica gel column (CHCl3-MeOH 9 : 1 ! 2 : 1). The methyl ester (MALDI-TOF
MS: m/z calcd for C30H44NaO15S ([M þ Na]þ): 699.2. Found 698.6) was hydrolyzed
on the column to the corresponding acid (MALDI-TOF MS: Calcd for C29H42NaO15S
([M þ Na]þ): 685.21, found 686.38). A mixture of this residue and benzyl bromide
(200 mL, 1.7 mmol) in dry DMSO (5 mL) was stirred at 158C. NaH (60% dispersion
in oil, 67 mg, 1.7 mmol) was added. After 30 min more BnBr (200 mL, 1.7 mmol)

414
Alpe, Oscarson, and Svahnberg
and NaH (1 mg, 3.0 mmol) were added and the reaction was stirred for 1 h at rt. The
reaction was quenched by the addition of toluene (165 mL) and water (55 mL).
The organic layer was separated, washed with HOAc (5% aqueous) and brine, dried,
and concentrated. Silica gel chromatography (toluene-EtOAc 9 : 1) gave 11 (35 mg,
24% in two steps) together with 8 (19 mg, 17%). 11: [a]D þ27 (c 1.0, CHCl3); NMR
13
data: C, d 15.0 (CH3CH2S), 25.5 (CH3CH2S), 62.2–84.2 (C-1-6, C-20-50, C-200-500,
CH2CHCH2O, PhCH2 ꢀ 8), 102.6, 103.3 (C-10,100), 116.9 (CH2CHCH2O), 127.4–129.1
(aromatic C), 135.2 (CH2CHCH2O), 137.9–138.7 (ipso-Bn), 167.9 (COOBn); MALDI-
TOF MS: m/z calcd for C78H84NaO15S ([M þ Na]þ): 1315.5. Found: 1315.5.
Anal. Calcd for C78H84O15S: C, 72.42; H, 6.57. Found: C, 72.19; H, 6.70.
2-Azidoethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-
(4,6-di-O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyra-
noside (14). A solution of 8 (26 mg, 26 mmol) and 12 (13 mg, 25 mmol) in dry diethyl ether
˚
(5 mL) containing powdered 4 A molecular sieves was stirred at rt in an argon atmosphere
for 30 min. DMTST (26 mg, 0.10 mmol) was added to the mixture and the stirring was
continued for 30 min. After neutralization with NEt3, the mixture was filtered through
Celite and concentrated. Purification by silica gel chromatography (toluene-EtOAc 9 : 1)
gave 2-azidoethyl (benzyl 2,3,4-tri-O-benzyl-b-D-glucopyranosyluronate)-(1 ! 2)-(3-
O-allyl-4,6-di-O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-D-mannopyra-
noside (13, 29 mg, 20 mmol, 81%). NMR data: 13C, d 50.3 (N3CH2CH2O), 66.6–83.5
(C-2-6, C-20-50, C-200-500, CH2CHCH2O, PhCH2 ꢀ 9, N3CH2CH2O), 97.9, 99.6 (C-1,10),
103.2 (C-100), 117.2 (CH2CHCH2O), 126.5–129.0 (aromatic C), 135.0 (CH2CHCH2O),
138.1–139.1 (ipso-Bn), 167.8 (COOBn). MALDI-TOF MS: m/z calcd for
C86H91NaO17 ([M þ Na]þ): 1460.6. Found 1459.7. PdCl2 (60%, 6 mg, 20 mmol) was
added to a solution of 13 (29 mg, 20 mmol) in EtOH : MeOH (1 : 1, 4 mL). After 30 min
more PdCl2 (10 mg, 34 mmol) was added. The mixture was stirred for 1 h, then filtered
through Celite, concentrated, and purified on a silica gel column (toluene-EtOAc 10 : 1)
to give 14 (21 mg, 15 mmol, 74%); [a]D þ17 (c 1.0, CHCl3); NMR data: 13C, d 50.3
(N3CH2CH2O), 66.6–83.0 (C-2-6, C-20-50, C-200-500, PhCH2 ꢀ 9, N3CH2CH2O), 98.0,
100.3 (C-1,10), 103.4 (C-100), 126.6–128.8 (aromatic C), 137.8–139.0 (ipso-Bn), 168.0
(COOBn); MALDI-TOF MS: m/z calcd for C83H87NaO17 ([M þ Na]þ): 1420.6. Found
1419.6.
Anal. Calcd for C83H87N3O17: C, 71.28; H, 6.27. Found: C, 71.02; H, 6.50.
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)-[(benzyl 2,3,4-tri-O-benzyl-b-D-glucopyra-
nosyluronate)-(1 ! 2)]-(4,6-di-O-benzyl-a-D-mannopyranosyl)-(1 ! 3)-2,4,6-tri-O-
benzyl-a-D-mannopyranoside (16). A solution of 15[8] (19 mg, 23 mmol) and 14 (16 mg,
˚
11 mmol) in dry diethyl ether (4 mL) containing powdered 4 A molecular sieves was stirred
at rt in an argon atmosphere for 30 min. DMTST (12 mg, 46 mmol) was added to the
mixture and the stirring was continued for 2 h. After neutralization with NEt3, the
mixture was filtered through Celite and concentrated. Purification by silica gel chromato-
graphy (toluene-EtOAc 9 : 1) gave 16 (22 mg, 10 mmol, 88%); NMR data: 13C, d 50.4
(N3CH2CH2O), 63.7–83.4 (C-2-6, C-20-50, C-200-500, C-2000-5000, C-20000-50000
,
CH2CHCH2O, PhCH2 ꢀ 14, N3CH2CH2O), 97.9, 99.9, 100.6 (C-1,10,1000), 102.6, 103.5
(C-100,10000), 116.6 (CH2CHCH2O), 126.5–129.0 (aromatic C), 135.3 (CH2CHCH2O),
138.0–139.0 (ipso-Bn), 167.7 (COOBn). MALDI-TOF MS: m/z calcd for
C
132H139N3NaO26 ([M þ Na]þ): 2204.9. Found: 2203.6.

Synthesis of Cryptococcus neoformans Structures
ACKNOWLEDGMENTS
415
Financial support from the Swedish Foundation for Strategic Research (the “Glyco-
conjugates in Biological Systems” programme) 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 in 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 Crypto-
coccus neoformans factor sera by enzyme-linked immunosorbent assay. Infect.
Immun. 1995, 63, 1810–1819.
8. Alpe, M.; Oscarson, S.; Svahnberg, P. Synthesis of Cryptococcus neoformans
capsular polysaccharide structures. Part IV: Construction of thioglycoside donor
blocks and their subsequent assembly. J. Carbohydr. Chem. 2003, 22, 565–577.
¨
9. Lonn, H. Synthesis of a disaccharide component of the capsular polysaccharide
antigen of Streptococcus pneumoniae serotype 1. Carbohydr. Res. 1984, 132, 39–44.
10. Alpe, M. Synthesis of oligosaccharides related to the capsular polysaccharides of
Streptococcus pneumoniae serotype 9 and of Cryptococcus neoformans. Stockholm
University, 2003; Doctoral Thesis.
11. Svahnberg, P. Synthesis of oligosaccharides containing deoxy functions and uronic
acids. Stockholm University, 2001; Doctoral Thesis.
12. Fischer, B.; Nudelman, A.; Ruse, M.; Herzig, J.; Gottlieb, H.E. A novel method for
stereoselective glucuronidation. J. Org. Chem. 1984, 49, 4988–4993.
13. Koto, S.; Miura, T.; Hirooka, M.; Tomaru, A.; Iida, M.; Kanemitsu, M.; Takenaka, K.;
Masuzawa, S.; Miyaji, S.; Kuroyanagi, N.; Yagishita, M.; Zen, S.; Yago, K.;
Tomonaga, F. Stereoselctive syntheses of a-glucuronides using dehydrative glycosy-
lation. Bull. Chem. Soc. Jpn. 1996, 69, 3247–3259.
14. Ek, M.; Garegg, P.J.; Hultberg, H.; Oscarson, S. Reductive ring openings of carbo-
hydrate benzylidene acetals using borane-trimethylamine and aluminium chloride.
Regioselectivity and solvent dependence. J. Carbohydr. Chem. 1983, 2, 305–311.
15. Fletcher, H.G.; Hudson, C.S. 1,5-anhydro-xylitol. J. Am. Chem. Soc. 1947, 69,
921–924.
16. 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.

416
Alpe, Oscarson, and Svahnberg
17. Olsson, L. Synthesis of oligosaccharides related to the capsular polysaccharide of
Cryptococcus neoformans; Stockholm University, 1996; Doctoral Thesis.
18. 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 exopolysac-
charide from Cryptococcus neoformans serovar D. Bioorg. Med. Chem. 1996, 4,
1819–1832.
19. 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.
Received June 15, 2004
Accepted August 25, 2004