Synthesis of tetra- and pentasaccharides corresponding to the capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N and 9A

Article abstract of DOI:10.1016/S0008-6215(03)00271-4

Two tetrasaccharides, α-D-GlcAp-(1→3)-α-D-Galp-(1→3)- β-D-ManpNAc-(1→4)-β-D-Glcp and α-D-GlcAp-(1→3)- α-D-Glcp-(1→3)-β-D-ManpNAc-(1→4)-β-D-Glcp (protected form), and a pentasaccharide, α-D-Glcp-(1→4)-α-D-GlcAp- (1→3)-α-D-Galp-(1→3)-β-D-ManpNAc-(1→4)-β-D-Glcp have been synthesised from 2-aminoethyl glycoside trisaccharide acceptors in a linear approach via consecutive α-glycosylations. Ethyl thioglycosides were used as glycosyl donors and DMTST in Et₂O or NIS/TfOH in CH ₂Cl₂ were employed as promoters.

Full text of DOI:10.1016/S0008-6215(03)00271-4

Carbohydrate Research 338 (2003) 2605ꢀ2609
/
www.elsevier.com/locate/carres
Synthesis of tetra- and pentasaccharides corresponding to the
capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N
and 9A
Mia Alpe, Stefan Oscarson*
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 19 March 2003; received in revised form 19 May 2003; accepted 21 May 2003
Abstract
Two tetrasaccharides, a-
D
-GlcAp-(10
/
3)-a-
-Glcp (protected form), and a pentasaccharide, a-
-Glcp have been synthesised from 2-aminoethyl glycoside trisaccharide acceptors in a linear
D
-Galp-(10
/
3)-b-
D
-ManpNAc-(10
/
4)-b-
D
-Glcp and a-
D
-GlcAp-(10
/
3)-a-
D
-Glcp-
(10
/
3)-b-
D
-ManpNAc-(10
/
4)-b-
4)-b-
D
D
-Glcp-(10
/
4)-a-
D
-GlcAp-(10
/
3)-a-
D-Galp-
(10
/
3)-b-
D
-ManpNAc-(10
/
D
approach via consecutive a-glycosylations. Ethyl thioglycosides were used as glycosyl donors and DMTST in Et2O or NIS/TfOH in
CH2Cl2 were employed as promoters.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Thioglycosides; Oligosaccharide synthesis; Glycoconjugate vaccines
1. Introduction
synthesised trisaccharide parts (residues IIIꢀI in Fig.
/
1) of the pentasaccharide repeating units of serotypes
9A, L and 9N.9 The trisaccharides were synthesised as
spacer glycosides, to allow conjugation to a carrier
protein, and the protection pattern was designed to
permit synthesis of larger structures. We now report the
continued synthesis, from these trisaccharide acceptors,
of tetra- and pentasaccharide structures, the latter
comprises the complete repeating unit of S. pneumoniae
type 9A.
The bacterium Streptococcus pneumoniae is a major
human pathogen.1 It is divided into serotypes, each
serotype corresponding to a unique structure of the
capsular polysaccharide (CPS) surrounding the bac-
teria.2 Serotype 9 is one of the most abundant serotypes
comprising several variations: 9A, 9L, 9N, and 9V (9A
with partial acetylation of the GlcA, ManNAc, and a-
Glc residues). These vary slightly in their CPS structure
(Fig. 1).3ꢀ6
Both polysaccharide and glycoconjugate vaccines
against S. pneumoniae are now commercial. The poly-
saccharide vaccine consists of 23 serotypes whereas the
conjugate vaccine contains seven serotypes, both, how-
ever, including serotype 9. Regarding polysaccharide
vaccines, there is a consensus that large structures are
needed, but with glycoconjugate vaccines there is a
debate on the saccharide size needed to give protec-
tion.7,8 To further investigate this we have earlier
2. Results and discussion
Since both tetra- and pentasaccharide structures were in
demand, a linear synthetic approach was applied.
Furthermore, we decided to use glucuronic acid donors
in the first coupling in spite of the their well-known low
reactivity both as donors and acceptors,10,11 since we
considered oxidation in high yield at the tetra- or
pentasaccharide level an even larger problem. Both
elongations, the introduction of an a-glucuronic acid
moiety as well as the subsequent introduction of an a-
glucose residue, were met with severe difficulties,
especially considering the stereochemical outcome.
* Corresponding author. Tel.: ꢁ
154908.
E-mail address: s.oscarson@organ.su.se (S. Oscarson).
/
46-8-162480; fax: ꢁ46-8-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00271-4

2606
M. Alpe, S. Oscarson / Carbohydrate Research 338 (2003) 2605ꢀ2609
/
Fig. 1. Repeating units of S. pneumoniae type 9 CPS.
Monochloroacetylation of the known ethyl thioglyco-
side 112 afforded 2 (Scheme 1). Removal of the trityl
10 with a free spacer amino group ready for conjuga-
tion.
Glycosidation of acceptor 9 with glucosyl donor 11,15
to afford a-linked pentasaccharides corresponding to
the type 9A repeating unit, was not straightforward
either (Scheme 3). As found also by others,11 the
stereoselectivity in a-glycosylations of this position is
low. Halide-assisted glycosylation conditions16 could
not be used due to the low reactivity of the 4-OH. The
best conditions were once again DMTST in diethyl
ether, which gave the pentasaccharide in 69% yield (2:1
a/b) and after chromatography the a-linked derivative
12 in 45% yield.
group followed by a two-step oxidation, a Pfitznerꢀ
/
Moffatt oxidation13 to the aldehyde and a subsequent
pyridine dichromate (PDC) oxidation in the presence of
methanol,14 afforded the methyl glucuronate donor 4 in
66% yield.
Our earlier experiences with thioglycoside donors in
a-glucuronosylations had shown that dimethyl(methyl-
thio)sulfonium trifluoromethanesulfonate (DMTST) in
diethyl ether gave preferentially the a-glycoside, whereas
N-iodosuccinimide (NIS)/TfOH in CH2Cl2 gave mainly
the b-linked glucuronate in spite of the absence of
participating groups.12 Consequently, when we started
to couple donor 4 with acceptor 59 to give a 9A,L type
tetrasaccharide, DMTST was tried as promoter. How-
ever, this gave surprisingly mainly the b-linked tetra-
saccharide. Several other conditions were tried and
finally it was found that instead NIS/TfOH in CH2Cl2
gave the best results considering stereoselectivity and
yield, 81% of a 2:1 a/b-mixture (Scheme 2). The
stereoisomers were easily separated to give the a-linked
tetrasaccharide 7 in 57% yield. The same conditions
were then tried for the coupling of donor 4 to the
glucosyl trisaccharide acceptor 69 for formation of type
9N structures, which resulted in the exclusive formation
of the b-linked tetrasaccharide. A return to the original
‘a-coupling’ conditions, i.e., DMTST in diethyl ether,
with this acceptor gave the expected outcome, i.e.,
mainly a-glucuronosylation, albeit with a low selectivity,
70% of a 3:2 a/b-mixture. Once more, separation of the
isomers was uncomplicated to yield the a-linked tetra-
saccharide 8 in 43% yield.
One-step deprotection of 12 through hydrogenolysis
then gave the pentasaccharide repeating unit of S.
pneumoniae type 9A 13 as its methyl ester and equipped
with an aminospacer for conjugation. 1H proton signals
Subsequent removal of the chloroacetyl group in 7
gave the acceptor 9 (76%), hydrogenolysis of which gave
the deprotected type 9A,L methyl ester tetrasaccharide
Scheme 1. (a) ClAcCl, pyridineꢀ
/
CH2Cl2; (b) pTsOH, CHCl3ꢀ
/
Scheme 2. (a) NISꢀ
/
TfOH, CH2Cl2 for 4ꢁ5; DMTST, Et2O
/
MeOH; (c) 1. TFA, DIC, Me2SO, pyridine, 2. PDC, MeOH.
for 4ꢁ6; (b) hydrazine acetate, MeOH; (c) H2, Pd(OH)2/C.
/

M. Alpe, S. Oscarson / Carbohydrate Research 338 (2003) 2605ꢀ
/2609
2607
temperature (rt), then diluted with toluene, concen-
trated, and purified by silica gel chromatography (15:1
tolueneꢀ/EtOAc) to give ethyl 2,3-di-O-benzyl-4-O-
chloroacetyl-6-O-triphenylmethyl-1-thio-b-
D
-glucopyr-
anoside (2, 7.9 g, 97%); 13C NMR (CDCl3): d 15.6
(SCH2CH3), 25.0 (SCH2CH3), 40.6 (CH2Cl), 62.8, 72.0,
75.6, 75.9, 77.7, 82.1, 83.9, 85.0, 86.9 (C-1-6, PhCH2O,
C(Ph)3), 127.3ꢀ
pound 2 (7.9 g, 11 mmol) was dissolved in 2:1 CHCl3ꢀ
/
143.8 (aromatic C), 165.8 (Cꢀ
/
O). Com-
/
MeOH and the pH was adjusted to 1 (pH-indicator
paper) by addition of p-toluenesulfonic acid. After 2 h,
the mixture was washed successively with water and satd
aq NaHCO3, dried and concentrated. Silica gel chro-
matography (4:1 tolueneꢀ
benzyl-4-O-chloroacetyl-1-thio-b-
4.3 g, 81%); 13C NMR (CDCl3): d 15.1 (SCH2CH3),
25.1 (SCH2CH3), 40.4 (CH2Cl), 61.8, 71.9, 75.5, 75.7,
/
EtOAc) gave ethyl 2,3-di-O-
D
-glucopyranoside (3,
77.9, 81.5, 83.4, 85.2 (C-1-6, PhCH2O), 127.9ꢀ
/138.2
(aromatic C), 166.6 (CꢀO). Compound 3 (1.3 g, 2.7
/
mmol) was dissolved in Me2SO (30 mL) at rt. To the
solution was added Py (220 mL, 2.7 mmol), trifluoro-
acetic acid (102 mL, 1.4 mmol), and diisopropylcarbo-
diimide (DIC) (1.50 mL, 9.6 mmol), and the reaction
was stirred overnight. The mixture was diluted with
toluene and washed with satd aq NaHCO3. After drying
and concentration of the organic phase the obtained
crude aldehyde was dried under diminished pressure and
used for the next oxidation without further purification.
The crude aldehyde was dissolved in DMF (25 mL)
containing MeOH (660 mL) and cooled on ice for 30 min
in a vessel covered to exclude light. PDC (6.0 g, 16.0
mmol) was added in one portion. The mixture was
allowed to attain rt and react overnight and was then
diluted with toluene, washed with water, dried and
Scheme 3.
at 5.39 (d, J 3.6 Hz, H-1V), 5.31 (d, J 3.4 Hz, H-1III),
and 5.14 (d, J 3.2 Hz, H-1IV) ppm clearly proves the a-
configurations of the newly synthesised linkages. The
corresponding protons in the native polysaccharide
resonance at 5.42 (Glc), 5.30 (Gal), and 5.09 (GlcA)
ppm.17
3. Experimental
3.1. General methods
concentrated. Silica gel chromatography (9:1 tolueneꢀ
/
Thin-layer chromatography (TLC) was carried out on
E. Merck precoated 60 F254 plates using UV-light and/or
8% H2SO4 for visualization. Column chromatography
EtOAc) gave 4 (917 mg, 66%) as a white solid; [a]D
ꢃ
/
388 (c 1.0, CHCl3); 13C NMR (CDCl3): d 15.0
(SCH2CH3), 25.1 (SCH2CH3), 40.4 (CH2Cl), 52.9
(CH3O), 72.4, 75.5, 75.7, 75.9, 80.9, 82.8, 85.5 (C-1-5,
was performed on silica gel (0.040ꢀ
in the flash mode, if not otherwise stated. NMR spectra
were recorded in CDCl3 at 25 8C (internal Me4Si, dꢂ
0.00 ppm) or in D2O at 70 8C (internal acetone 13C dꢂ
/
0.063 mm, Amicon)
PhCH2O), 127.9ꢀ
O). Anal. Calcd for C25H29ClO7S: C, 59.0; H, 5.7.
/
138.0 (aromatic C), 166.1, 167.4 (Cꢀ
/
/
/
Found: C, 59.1; H, 5.9.
1
31.0 ppm, H dꢂ
/
2.21 ppm) on a Varian 300 or 400
TOF mass spectra were
MHz instrument. MALDIꢀ
/
3.3. 2-(N-Benzyloxycarbonyl)-aminoethyl (methyl 2,3-di-
recorded on a Bruker Biflex III instrument using
2?,4?,6?-trihydroxyacetophenone trihydrate (THAP) as
matrix. Organic phases were dried over Na2SO4 before
concentration, which was performed under diminished
pressure.
O-benzyl-4-O-chloroacetyl-a-
(103)-(2,4,6-tri-O-benzyl-a-
(2-acetamido-4,6-O-benzylidene-2-deoxy-b-
mannopyranosyl)-(104)-2,3,6-tri-O-benzyl-b-
glucopyranoside (8)
D
-glucopyranosyluronate)-
/
D-glucopyranosyl)-(103)-
/
D-
/
D-
3.2. Methyl (ethyl 2,3-di-O-benzyl-4-O-chloroacetyl-1-
thio-b-D-glucopyranosid)uronate (4)
A solution of 4 (60 mg, 118 mmol) and 69 (80 mg,
59 mmol) in dry Et2O (5 mL) containing powdered
molecular sieves (4 A) was stirred at rt in an Ar
˚
Chloroacetyl chloride (1.8 mL, 23 mmol) was added to a
solution of 112 (7.3 g, 11 mmol) in 15:1 CH2Cl2ꢀ
Py at
0 8C. The reaction mixture was stirred for 2 h at room
atmosphere for 1 h. To the mixture was added DMTST
(122 mg, 472 mmol) and the stirring was continued
overnight. After neutralization with Et3N, the mixture
/

2608
M. Alpe, S. Oscarson / Carbohydrate Research 338 (2003) 2605ꢀ2609
/
was filtered through Celite and concentrated. Silica gel
chromatography (1:1 tolueneꢀEtOAc) of the residue
yielded 8 as an a/b-mixture (74 mg, 41.1 mmol, 70%).
Repeated chromatography (2 columns, not flash-mode;
yielded 2-(N-benzyloxycarbonyl)-aminoethyl (methyl
2,3-di-O-benzyl-a- -glucopyranosyluronate)-(103)-
(2,4,6-tri-O-benzyl-a- -galactopyranosyl)-(103)-(2-
acetamido-4,6-O-benzylidene-2-deoxy-b- -mannopyra-
-glucopyranoside
/
D
/
D
/
D
1:1 tolueneꢀ
25.6 mmol, 43%). [a]D
NMR data (CDCl3): d 52.6 (C-2II, CH3O), 66.5ꢀ
(C-2Iꢀ6I, 3IIꢀ6II, IIIꢀ6III, IVꢀ
/
EtOAc) afforded the pure a anomer (46 mg,
nosyl)-(10
/
4)-2,3,6-tri-O-benzyl-b-
D
ꢁ
/
188 (c 1.0, CHCl3); Selected 13
C
83.5
(9, 37 mg, 21.6 mmol, 76%); 13C NMR (CDCl3): d 23.1
(CH3CON), 41.3(CH2NHCOOBn), 52.0, 52.3 (CH3O,
C-2II), 66.5, 66.7, 68.2, 68.6, 69.6, 69.8, 70.9, 72.0, 72.6,
73.2, 73.5, 73.6, 73.9, 74.4, 74.9, 75.0, 75.1, 75.3, 75.8,
/
/
/
2
/
2
/
5IV, OCH2CH2N,
PhCH2O), 96.4, 97.4 (C-1III, 1IV), 100.0, 102.7, 104.1
(C-1I, 1II, benzylidene), 126.5ꢀ
139.1 (aromatic C),
156.7, 166.3, 166.5, 170.6 (CꢀO).; b anomer: d 96.9
(C-1III), 99.9, 102.1, 102.4, 104.1 (C-1I, 1II, 1IV, benzyl-
idene). MALDIꢀTOFMS: Calcd for C102H109ClN2-
NaO25 ([Mꢁ
Na]ꢁ): 1819.7. Found 1820.2.
/
77.6, 78.8, 79.8, 80.6, 81.8, 83.1 (C-2Iꢀ
6III, 2IVꢀ5IV, OCH2CH2N, PhCH2O), 96.5, 97.2 (C-1III
1IV), 99.5, 101.9, 103.8 (C-1I, 1II, benzylidene), 125.3ꢀ
139.1 (aromatic C), 156.4 (NHCOOBn), 170.6, 171.2
(CꢀO). MALDIꢀTOFMS: Calcd for C100H109N2NaO24
([Mꢁ
Na]ꢁ): 1743.7. Found: 1743.1. To a solution of 9
(25 mg, 14.5 mmol) dissolved in 3:2 EtOHꢀAcOH (5
/
6I, 3IIꢀ
/
6II, 2IIIꢀ
/
/
/
,
/
/
/
/
/
/
3.4. 2-Aminoethyl (methyl a-
D
-glucopyranosyluronate)-
-galactopyranosyl-(103)-2-acetamido-2-
-mannopyranosyl-(104)-b- -glucopyranoside
/
(10
/
3)-a-
D
/
mL) was added palladium hydroxide on activated
charcoal and the mixture was hydrogenolyzed at 0.69
MPa for 72 h. Additional catalyst was added after 24
and 48 h. The mixture was centrifuged and the pellets
were washed once with EtOH. The supernatants were
combined and concentrated. Purification of the residue
on a Bio-Gel P-2 column gave, after freeze drying, 10 (8
deoxy-b-
(10)
D
/
D
A mixture of 4 (64 mg, 0.13 mmol) and 59 (100 mg, 74
mmol) in dry CH2Cl2 containing powdered molecular
˚
sieves (4 A) was stirred under Ar at rt for 1 h. The
solution was cooled to ꢃ30 8C, NIS (28 mg, 0.13 mmol)
and TfOH (3 mL, 37 mmol) were added and the mixture
was stirred for 5 h at ꢃ30 8C. The mixture was
/
mg, 10.3 mmol, 71%); [a]D
ꢁ398 (c 0.8, water); Selected
/
NMR data (D2O): 13C, d 95.9 (C-1IV), 100.3 (C-1II),
1
/
101.2 (C-1III), 103.0 (C-1I); H, d 4.52 (d, 1 H, J1,2 8.1
neutralized with Et3N and filtered through Celite. The
filtrate was washed with Na2S2O3 (10% aq), NaHCO3
(aq satd) and water, dried and concentrated. Purifica-
Hz, H-1I), 4.90 (s, 1 H, H-1II), 5.17 (d, 1 H, J1,2 3.7 Hz,
H-1IV), 5.32 (d, 1 H, J1,2 3.8 Hz, H-1III). MALDIꢀ
/
TOFMS: Calcd for C29H50N2NaO22 ([Mꢁ
Na]ꢁ):
801.3. Found: 801.1.
/
tion by silica gel chromatography (1:1 tolueneꢀ
gave 2-(N-benzyloxycarbonyl)-aminoethyl (methyl 2,3-
di-O-benzyl-4-O-chloroacetyl- -glucopyranosyluro-
nate)-(103)-(2,4,6-tri-O-benzyl-a- -galactopyranosyl)-
(103)-(2-acetamido-4,6-O-benzylidene-2-deoxy-b-
mannopyranosyl)-(10 -gluco-
/EtOAc)
D
3.5. 2-Aminoethyl a-
D
-glucopyranosyl-(10
3)-a- -galactopyranosyl-
3)-2-acetamido-2-deoxy-b-
-mannopyranosyl-(10
D-glucopyranoside (13)
/
4)-(methyl a-
/
D
D
-glucopyranosyluronate)-(10
/
D
/
D
-
(10
/
D
/
/
4)-2,3,6-tri-O-benzyl-b-
D
4)-b-
pyranoside as an a/b-mixture (107 mg, 60 mmol, 81%).
Repeated chromatography (2 columns, not flash-mode;
EtOAc) afforded the pure a anomer (7, 76
A mixture of 1115 (13 mg, 22.1 mmol) and 9 (19 mg, 11.0
mmol) in dry Et2O (5 mL) containing powdered mole-
cular sieves (4 A) was stirred at rt in an Ar atmosphere
1:1 tolueneꢀ
mg, 57%); [a]D
/
188 (c 1.0, CHCl3); 13C NMR
˚
ꢁ
/
(CDCl3): d 23.3 (CH3CON), 40.6, 41.6 (CH2Cl,
CH2NHCOOBn), 52.5 (C-2II, CH3O), 66.7, 67.0, 68.4,
68.9, 69.4, 69.8, 72.4, 73.2, 73.4, 73.5, 73.7, 74.3, 74.6,
75.1, 75.2, 75.5, 75.9, 76.1, 78.5, 79.3, 80.1, 82.0, 83.3 (C-
for 1 h. To the mixture was added DMTST (11 mg, 44.1
mmol) and the stirring was continued for 6 h. After
neutralization with Et3N, the mixture was filtered
through Celite and concentrated. The residue was
2I ꢀ
PhCH2O), 96.6, 96.8 (C-1III, 1IV), 99.7, 102.2, 104.0
(C-1, 1?, benzylidene), 126.4ꢀ139.3 (aromatic C), 156.7
(NHCOOBn), 166.4, 169.1 and 170.9 (CꢀO); b anomer
(selected data): d 97.1 (C-1III), 99.4, 102.1, 103.7, 103.8
(C-1I, 1II, 1IV, benzylidene). MALDIꢀ
TOFMS: Calcd
for C102H109ClN2NaO25 ([Mꢁ
Na]ꢁ): 1819.7. Found:
/
6I, 3II ꢀ
/
6II, 2III ꢀ
/
6III, 2IV ꢀ
/
5IV, OC H2CH2N,
purified on a silica gel column (2:1 tolueneꢀ
yield 2-(N-benzyloxycarbonyl)-aminoethyl (2,3,4,6-tet-
ra-O-benzyl- 4)-(methyl 2,3-di-
-glucopyranosyl)-(10
-glucopyranosyluronate)-(103)-(2,4,6-tri-
-galactopyranosyl)-(103)-(2-acetamido-
-mannopyranosyl)-(10
-glucopyranoside as an a/b-
mixture (17 mg, 7.6 mmol, 69%). Repeated chromato-
graphy (2 columns, not flash-mode; 2:1 tolueneꢀEtOAc)
/
EtOAc) to
/
D
/
/
O-benzyl-a-
D
/
,O-benzyl-a-
D
/
/
4,6-O-benzylidene-2-deoxy-b-
4)-2,3,6-tri-O-benzyl-b-
D
/
/
D
1820.4. Hydrazine acetate (51 mg, 0.6 mmol) was added
to a solution of 7 (51 mg, 0.3 mmol) in MeOH (5 mL)
and the mixture was stirred overnight. Additional
hydrazine acetate (25 mg, 14 mmol) was added and after
an additional 2 h the mixture was concentrated. Silica
/
afforded the pure a anomer (12, 11 mg, 4.9 mmol, 45%);
Selected NMR data: 1H, d 5.06 (d, 1 H, 3.5 Hz), 5.42 (d,
1 H, 3.5 Hz), 5.53 (d, 1 H, 3.3 Hz). MALDIꢀ
/
TOFMS:
Na]ꢁ): 2266.0.
gel chromatography (1:1 tolueneꢀ/EtOAc) of the residue
Calcd for C134H142N2NaO29 ([Mꢁ
/

M. Alpe, S. Oscarson / Carbohydrate Research 338 (2003) 2605ꢀ
/2609
2609
2. Lindberg, B.; Kenne, L. In The Polysaccharides; Aspinall,
G. O., Ed.; Vol. 2; Academic Press: New York, 1995; pp
Found: 2266.6. To a solution of 12 (11 mg, 4.9 mmol)
dissolved in 3:2 EtOHꢀAcOH (5 mL) was added
/
287ꢀ
3. Kamerling, J. P. Pneumococcal polysaccharides: A chemi-
cal view; In Ref. 1 pp. 81ꢀ114.
4. Jones, C.; Mulloy, B.; Wilson, A.; Dell, A.; Oates, J. E. J.
Chem. Soc., Perkin Trans. 1 1985, 1665ꢀ1673.
5. Richards, J. C.; Perry, M. B.; Kniskern, P. J. Can. J.
Biochem. 1984, 62, 1309ꢀ1320.
6. Perry, M. B.; Daoust, V.; Carlo, D. J. Can. J. Biochem.
1981, 59, 524ꢀ533.
7. Laferrie`re, C. A.; Sood, R. K.; deMuys, J.-M.; Michon, F.;
Jennings, H. J. Vaccine 1997, 15, 179ꢀ186.
/
363.
palladium hydroxide on activated charcoal and the
mixture was hydrogenolyzed at 0.69 MPa for 72 h.
Additional catalyst was added after 48 h. The mixture
was centrifuged and the pellets were washed once with
EtOH. The supernatants were combined and concen-
trated. Purification of the residue on a Bio-Gel P-2
column gave, after freeze-drying, 13 (4 mg, 3.72 mmol,
/
/
/
76%). [a]D
ꢁ738 (c 0.5, water). Selected NMR data
/
/
(D2O): H, d 4.49 (d, 1 H, J1,2 7.9 Hz, H-1I), 4.88 (s, 1
H, H-1II), 5.14 (d, 1 H, J1,2 3.2 Hz, H-1IV), 5.31 (d, 1 H,
J1,2 3.4 Hz, H-1III), 5.39 (d, 1 H, J1,2 3.6 Hz, H-1V).
1
/
8. Benaissa-Trouw, B.; Lefeber, D. J.; Kamerling, J. P.;
Vliegenthart, J. F. G.; Kraaijeveld, K.; Snippe, H. Infect.
MALDIꢀ
/
TOFMS: Calcd for C35H60N2NaO27 ([Mꢁ
/
Na]ꢁ): 963.3. Found: 963.3.
Immun. 2001, 69, 4698ꢀ
/
4701.
9. Alpe, M.; Oscarson, S. Carbohydr. Res. 2002, 337, 1715ꢀ
/
1722.
10. Stachulski, A. V.; Jenkins, G. N. Natural Prod. Rep. 1998,
Acknowledgements
15, 173ꢀ
11. Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P.
Angew. Chem. Int. Ed. 2002, 41, 2128ꢀ2131.
12. Oscarson, S.; Svahnberg, P. J. Chem. Soc., Perkin Trans. 1
2001, 873ꢀ879.
13. Pfitzner, K. E.; Moffatt, J. G. J. Am. Chem. Soc. 1963, 85,
3027ꢀ3028.
14. O’Connor, B.; Just, B. Tetrahedron Lett. 1987, 28, 3235ꢀ
3236.
15. Andersson, F.; Fugedi, P.; Garegg, P. J.; Nashed, M.
/
186.
/
Financial support from EU (Contract Number BIO 4
CT 960158), from the Swedish Foundation for Strategic
Research (the ‘Glycoconjugates in Biological Systems’
programme) and from the Swedish Natural Science
Research Council are gratefully acknowledged.
/
/
/
¨
References
Tetrahedron Lett. 1986, 27, 3919ꢀ
16. Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K. J.
Am. Chem. Soc. 1975, 97, 4056ꢀ4062.
17. Rutherford, T. J.; Jones, C.; Davies, D. B.; Elliott, A. C.
Carbohydr. Res. 1994, 265, 97ꢀ111.
/
3922.
1. Streptococcus pneumoniae; Thomasz, A. (Ed.); Molecular
Biology & Mechanisms of Disease, Mary Ann Liebert,
Inc., New York, 2000.
/
/