Synthesis of oligosaccharides related to the repeating unit of the capsular polysaccharide from Streptococcus pneumoniae type 37

Article abstract of DOI:10.1016/j.carres.2004.11.004

A tetra- and a pentasaccharide were synthesized as analogues to the structure of the Streptococcus pneumoniae type 37 capsular polysaccharide, a homopolymer with a disaccharide-repeating unit of →3)[β-d-Glcp- (1→2)]-β-d-Glcp-(1→. Synthesis of the tetrasaccharide employed a β-(1→2)-diglycosylation of a β-(1→3)-linked disaccharide. Subsequently, the pentasaccharide was synthesized from a suitably protected tetrasaccharide derivative by a β-(1→3)-extension at O-3′. Steric crowding was found to be an important factor in the formation of the pentasaccharide.

Full text of DOI:10.1016/j.carres.2004.11.004

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 7–13
Rapid communication
Synthesis of oligosaccharides related to the repeating unit of the
capsular polysaccharide from Streptococcus pneumoniae type 37
E. Andreas Larsson, Mats Sjoberg and Goran Widmalm*
¨
¨
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 17 August 2004; received in revised form 21 September 2004; accepted 6 November 2004
Available online 25 November 2004
Abstract—A tetra- and a pentasaccharide were synthesized as analogues to the structure of the Streptococcus pneumoniae type 37
capsular polysaccharide, a homopolymer with a disaccharide-repeating unit of !3)[b-D-Glcp-(1!2)]-b-D-Glcp-(1!. Synthesis of
the tetrasaccharide employed a b-(1!2)-diglycosylation of a b-(1!3)-linked disaccharide. Subsequently, the pentasaccharide was
synthesized from a suitably protected tetrasaccharide derivative by a b-(1!3)-extension at O-30. Steric crowding was found to be
an important factor in the formation of the pentasaccharide.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Bacterial polysaccharide; Glycosylation; Branched structure; Steric
1. Introduction
sons for obtaining more information on these complex
biopolymers, their structures and their properties.
Streptococcus pneumoniae, however, is a major cause
of acute respiratory infections worldwide.2 Recent
developments using synthetic oligosaccharide–protein
conjugates are promising, since protective antibodies
against specific pneumococci serotypes can be induced
upon immunization.3,4 Thus, the approach may lead to
effective vaccines against pneumococci.
Carbohydrate polymers constitute part of the cell wall
of bacteria as well as the coating at the bacterial cell sur-
face as lipopolysaccharides (LPS) and/or capsular
polysaccharides (CPS).1 These polysaccharides are
important for the bacteria since they can facilitate pro-
tection against phagocytosis and increase survival in a
foreign host, for example, man. Parts of a polysacchar-
ide form epitopes, that is, three-dimensional structures
recognized by other molecules, in particular antibodies
and lectins. These carbohydrate polymers may be simi-
lar to endogenous carbohydrate structures on cells,
thereby evading the protective immune system of the
host. The CPS is commonly also a most important viru-
lence factor, meaning that without it the pathogenÕs
capability to infect is severely reduced. In addition, the
polysaccharides confer the bacterium with suitable rheo-
logical properties for its survival in different environ-
ments. It must be noted that, although a great many
bacteria are pathogenic, most are not a threat to man
and some are even beneficial. Thus, there are several rea-
2. Results and discussion
The CPS from the bacterium S. pneumoniae type 37
(S37) is the only homopolymer reported for the pneu-
mococcus species and it consists of repeating units with
two glucosyl residues having the structure: !3)[b-D-
Glcp-(1!2)]-b-D-Glcp-(1!.5 It is anticipated that this
branched type of polymer will form a crowded three-
dimensional structure. Interestingly, it has been shown
that only one glycosyl transferase is needed in the bio-
synthesis of this CPS, even though two residues in a dif-
ferent setting are to be synthesized.6 The smallest
oligosaccharide possibly being a relevant model for the
polymer would be a tetrasaccharide. Whether this is
*
Corresponding author. Tel.: +46 8 16 37 42; fax: +46 8 15 49 08;
e-mail: gw@organ.su.se
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.11.004

8
E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
sufficient or if higher analogues such as hexa- or octasac-
charides are necessary to describe the three-dimensional
structure is of fundamental interest and it should be pos-
Ph
O
Ph
O
O
O
O
O
HO
SEt
BnO
+
BzO
AcO
OMe
1
sible by H NMR analysis to ascertain this issue, prior
3
4
to analysis of the conformational dynamics of an appro-
priate oligosaccharide. Since also short oligomers of
polysaccharides can generate a sufficient antibody re-
sponse,7 an efficient synthesis of the S. pneumoniae type
37 CPS analogues could be used to prepare oligosacchar-
ides for conjugation to proteins, and thus to be used as
vaccines. We have previously synthesized trisaccharides
with similar structures,8,9 and therefore set out to syn-
thesize oligosaccharides related to the repeating unit of
the CPS of S37.
Two structures related to the CPS were synthesized
(Fig. 1). At the reducing end of the oligosaccharides to
be synthesized we chose an a-D-Glc-OMe residue since
it provides a handle when analyzed by NMR spectro-
scopy. Our synthetic route to tetrasaccharide 2 first in-
volved formation of the b-(1!3)-linked disaccharide
corresponding to the backbone of the polymer. This
would allow us to introduce the two b-(1!2)-linked res-
idues in one step through a diglycosylation reaction with
the disaccharide acceptor. Moreover, it also seemed a
plausible strategy for the synthesis of larger analogues
as well. As glycosyl donor for the first glycosylation
we selected ethyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylid-
ene-1-thio-b-D-glucopyranoside 3.10 Methyl 2-O-benz-
oyl-4,6-O-benzylidene-a-D-glucopyranoside 411 was
used as the common reducing end acceptor for all deriv-
atives synthesized.
i
Ph
O
O
Ph
O
O
O
O
O
BnO
R2O
R1O
OMe
5
6
R1 = Ac, R2 = Bz (83%)
R1 = H, R2 = H (99%)
ii
Scheme 1. Formation of disaccharide acceptor 6. Reagents and
conditions: (i) NIS/TfOH, CH2Cl2, 25ꢁC, 0.5h; (ii) NaOMe, MeOH/
CH2Cl2 2/1, 25ꢁC, 2h.
the disaccharide diol acceptor 6 in 99% yield. Diglycosyl-
ation of 6 with thioglycosyl donors and NIS/TfOH or
DMTST14 activation resulted in poor yields due to
incomplete glycosylation forming a mixture of tri- and
tetrasaccharides. However, tetrasaccharide 8 could be
produced in 84% yield by a AgOTf-promoted15 glycosyl-
ation with 4.5equiv of 2,3,4,6-tetra-O-benzoyl-a-D-
glucosyl bromide 7 in the presence of sym-collidine at
ꢀ55ꢁC. The benzoyl groups were removed by treatment
of 8 with sodium methoxide in methanol. Catalytic
hydrogenolysis (5% Pd–C) in a mixture of methanol,
water and acetic acid removed the remaining protecting
groups yielding the deprotected tetrasaccharide 2 in 79%
yield (Scheme 2).
NIS/TfOH-promoted12 glycosylation of donor 3 in
CH2Cl2 at room temperature with acceptor 4 gave disac-
charide 5 in 83% yield (Scheme 1). The b-(1!3) glycos-
idic linkage can also be formed with the same donor and
promotor system but with methyl 4,6-O-benzylidene-
a-D-glucopyranoside13 as acceptor in a regioselective
manner in 73% yield. However, solubility problems
hampered the purification and rendered this shortcut
less practical when scaled up.
The 1H NMR spectrum of 2 showed extensive overlap
in the anomeric region for the two b-(1!2)-linked gluco-
syl residues. However, if the temperature was raised to
70ꢁC the anomeric signals could be resolved. In spite of
this, these conditions are not particularly suitable for fu-
1
Treatment of disaccharide 5 with methanolic sodium
methoxide removed the ester protecting groups yielding
ture investigations. In addition, the tetrasaccharide H
and 13C NMR chemical shifts also deviates considerably
C
1
B
A
B
A
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
O
O
O
HO
O
O
O
HO
OMe
OMe
O
O
O
O
HO
HO
2
HO
HO
OH
OH
OH
OH
OH
OH
OH
OH
HO
A'
HO
HO
HO
B'
Figure 1. Pentasaccharide 1 and tetrasaccharide 2.
B'
A'

E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
9
Ph
O
O
Ph
O
Ph
O
O
Ph
O
O
O
O
O
O
O
O
O
O
O
BnO
BnO
HO
OH
OMe
i
ii, iii
OMe
2
(79%)
6
+
O
O
BzO
OBz
BzO
OBz
OBz
O
BzO
BzO
OBz
OBz
BzO
(84%)
BzO
BzO
Br
8
7
iv, ii, v
AcO
AcO
AcO
R1O
O
O
O
AcO
O
O
OMe
O
O
AcO
OAc
AcO
OAc
OAc
OAc
AcO
AcO
9
R1 = Bn (73%)
iii
10 R1 = H (95%)
Scheme 2. Formation, modification and deprotection of the tetrasaccharide. Reagents and conditions: (i) AgOTf, CH2Cl2, ꢀ55ꢁC, 5h; (ii) NaOMe,
MeOH; (iii) H2/Pd–C; (iv) 90% aq TFA; (v) Ac2O, pyridine, DMAP, CH2Cl2.
from those observed in the native polymer, indicating
that a larger analogue is needed if a polymer-like tertiary
structure is to be present.
extent compatible with the conditions necessary to form
the corresponding tetrasaccharide. Instead, we decided
to exploit the protecting group pattern already present
in tetrasaccharide 8 (Scheme 2), that is, to use the
orthogonality of the benzyl ether.
Given the fact that the diglycosylation was successful,
a triglycosylation appeared to be an efficient route to a
hexasaccharide representing a trimer of the repeating
unit in the polymer. Hence, a trisaccharide triol acceptor
was successfully constructed by elongation at O-30,
based on an acceptor produced via the 30-O-p-meth-
oxybenzyl analogue of 5 followed by the removal of
the ester functionalities. The glycosylation of this sub-
stance with 7 was sluggish, producing mainly pentasac-
charides. Only a trace amount of the hexasaccharide
was formed according to MALDI-TOF MS. In addi-
tion, an O-benzylated trisaccharide triol acceptor lack-
ing all the acid labile protecting groups was also
produced, albeit without improving the outcome of the
glycosylation. The results of the glycosylations employ-
ing trisaccharide acceptors and applying a strategy sim-
ilar to that used to form the tetrasaccharide were not
encouraging. An alternative route with a different accep-
tor was therefore needed.
The 4,6-O-benzylidene acetal and benzoyl groups of 8
were removed using 90% aq TFA and sodium methox-
ide in methanol, respectively. Subsequent O-acetylation
was performed by Ac2O in pyridine to give 9 in 73%
over three steps. Initially, considering the superior sta-
bility of benzoyl esters as compared to acetyl groups
we synthesized the O-benzoylated analogue of 9. How-
ever, removal of the benzyl group turned out to be prob-
lematic. Treatment with H2/Pd–C at 110psi produced
only small amounts of the O-benzoylated acceptor.
Reactions involving heterogeneous catalysts are sensi-
tive to steric hindrance. A method that should be more
compatible with steric hindrance for removal of the O-
benzyl group by reaction with sodium bromate/sodium
dithionite in ethyl acetate/water,16 only partially solved
the problem since the yields were moderate. Even worse,
the formed acceptor did not work well in glycosylations,
presumably as a consequence of steric hindrance. To re-
duce the amount of possible steric interactions we sub-
stituted the benzoyl groups at the 4- and 6-positions
on the backbone glucosyl residues with O-acetyl groups,
yet the results were analogous to the O-benzoylated
With this in mind, it seemed reasonable to believe that
if we produced a tetrasaccharide carrying an orthogonal
protecting group at the O-30-position further elongation
should be possible. Neither 30-O-allyl nor p-meth-
oxybenzyl protected analogues of 6 were to any greater

10
E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
AcO
AcO
AcO
O
AcO
AcO
AcO
O
O
O
O
AcO
O
O
AcO
AcO
AcO
O
i
AcO
+
10
SEt
OMe
AcO
O
O
AcO
AcO
OAc
OAc
11
OAc
OAc
AcO
AcO
12 (58%)
ii
HO
HO
HO
HO
HO
O
O
O
HO
O
HO
O
O
O
HO
OMe
O
O
HO
HO
OH
OH
OH
OH
HO
1 (54%)
HO
˚
Scheme 3. Formation and deprotection of the pentasaccharide. Reagents and conditions: (i) NIS/TfOH, MS 4A, CH2Cl2, 25ꢁC; (ii) NaOMe,
MeOH.
derivative. However, if also the benzoyl groups at the
terminal glucosyl residues were exchanged for O-acetyl
groups, removal of the O-benzyl group from 9 by cata-
lytic hydrogenation succeed in giving tetrasaccharide
acceptor 10 (Scheme 2). More importantly, NIS/
TfOH-promoted glycosylation of 10 at room tempera-
ture with the per-O-acetylated thioglucosyl donor 1117
produced the per-O-acetylated pentasaccharide 12 in
58% yield (Scheme 3). Final deprotection by sodium
methoxide in methanol followed by purification via gel
permeation chromatography yielded 54% of pentasac-
charide 1. One may note that in this pentasaccharide
3. Experimental
3.1. General procedures
TLC was performed on Precoated Merck 60 F254 plates
and visualized with UV-light or 8% H2SO4. For column
chromatography Amicron silica gel 0.040–0.063mm was
used. NMR spectroscopy was performed on Varian 300,
400 and 600MHz spectrometers at 25ꢁC. Chemical
shifts are referenced to internal TMS (dH 0.00) and to
the solvent in CDCl3 (dC 77.23), to internal Sodium 3-
trimethylsilyl-propanoate-2,2,3,3-d4, (TSP) (dH 0.00)
and external dioxane in D2O (dC 67.4) for D2O
solutions.
For assignments of 1H and 13C resonances, the follow-
ing experiments were used: 13C APT, 1H,1H COSY,
1H,1H TOCSY (mixing time 30ms), 1H,13C HSQC,
1H,13C HMBC (mixing time 50ms). Backbone residues
are referred to as A, B and C, where A corresponds to
the reducing end and side chains are primed and denoted
by A0 and B0 in relation to their backbone residues.
MALDI-TOF MS was performed on a Bruker Biflex
III spectrometer in reflex mode with 20,40,60-tri-
hydroxyacetophenone (THAP) as a matrix. A typical
sample preparation from organic solvents employed
0.5lL of a very dilute solution of the sample applied
onto a crystal layer of THAP, whereas from water a
0.5lL sample was first premixed with 0.5lL of a 1/1
solution of H2O and a saturated solution of THAP in
acetone, followed by application to a crystal layer of
THAP.
1
the anomeric H chemical shift of residue B is closely
similar to the corresponding one in the backbone of
the CPS from S37 and that dH1 of B0 has changed com-
pared to tetrasaccharide 2 towards that observed for the
side-chain residue in the polymer. Recently, the EPS of
P. freudenreichii ssp. shermanii JS was shown to produce
a glucan with an identical disaccharide-repeating unit to
1
that of the CPS from S37.18 The reported H and 13C
NMR chemical shifts of the EPS will be useful in com-
parison to the synthesized oligosaccharides.
We have shown that reducing the size of the protect-
ing groups is essential for this system, also for the gluco-
syl units not directly associated with the site of
glycosylation. With the successful introduction of the
fifth glucosyl residue, this approach might allow the syn-
thesis of a hexasaccharide corresponding to a trimer of
the repeating unit of the CPS from S37. However, as the
oligosaccharide structure approaches that of the native
polymer, an increasing steric demand is to be expected.

E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
11
3.2. Experimental
Et3N. The solution was then filtered through a pad of
Celite and evaporated. The crude product was purified
by silica column chromatography (gradient, T!T/E:
2/1) to give an 84% yield of tetrasaccharide 8 (240mg,
0.135mmol). MALDI-MS: [M+Na]+ m/z 1802.4, ex-
pected 1802.6. 13C NMR: 55.7 (OMe), 61.8–81.4 (C-2–
C-6, OCH2Ph), 99.4, 100.6, 100.7, 100.8, 101.3, 101.4
(6 · acetal C), 126.1–138.6 (aromatic C), 165.2–166.2
(C@O).
3.2.1. Methyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-
b-D-glucopyranosyl-(1!3)-2-O-benzoyl-4,6-O-benzylidene-
a-D-glucopyranoside (5). A solution of methyl 2-O-
benzoyl-4,6-O-benzylidene-a-D-glucopyranoside (1.32g,
3.41mmol) and ethyl 2-O-acetyl-3-O-benzyl-4,6-O-benz-
ylidene-1-thio-b-D-glucopyranoside (1.67g, 3.75mmol)
dissolved in 12mL of CH2Cl2 under Argon atmosphere
˚
was pre-dried for 25min with crushed 4A molecular
sieves. NIS (0.915g, 4.09mmol) was added to the reac-
tion mixture in one portion. The reaction was initiated
by the addition of a catalytic amount of TfOH. The
reaction was followed by TLC (T/E: 3/1), which after
40min indicated that the starting materials had been
consumed. The reaction mixture was filtered directly
through a short plug of silica into a separating funnel
containing 100mL of aq Na2S2O3 and NaHCO3. The
organic phase was washed and separated, dried with
Na2SO4 and evaporated. The crude product was purified
by silica column chromatography (T/E: 6/1) to give 3
(2.18g, 2.84mmol) as a white solid in 83% yield. MAL-
DI-MS: [M+Na]+ m/z 791.7, expected 791.3. 13C NMR:
20.4 (Me), 55.6 (OMe), 62.9, 66.4, 68.9, 69.1, 72.8, 73.9,
74.3, 75.9, 78.8, 79.3, 81.3 (10 · C-2–C-6, OCH2Ph),
97.8, 101.3, 101.4, 101.6 (4 · acetal C), 126.2–133.7 (aro-
matic C), 166.0, 169.4 (2 · C@O).
3.2.4. Methyl [b-D-glucopyranosyl-(1!2)]-b-D-glucopyra-
nosyl-(1!3)-[b-D-glucopyranosyl-(1!2)]-a-D-glucopyra-
noside (2). To a solution of 8 (53mg, 0.030mmol) in
2mL MeOH/toluene: 3/1, 1M NaOMe was added until
the pH > 12. After 3h MALDI-MS showed only the O-
deacylated tetrasaccharide ([M+Na]+ m/z 969.4, ex-
pected 969.3). The reaction mixture was neutralized with
Dowex-(H+), filtered and subsequently evaporated to
dryness. The crude product was then directly dissolved
in 4mL of MeOH/HOAc/H2O: 6/1/1. A catalytic
amount of 5% Pd–C was added and the reaction vessel
transferred to a Parr apparatus and subjected to a
111psi of H2 for 20h. MALDI-MS confirmed complete
disappearance of the starting material. The reaction
mixture was filtered through a pad of Celite, concen-
trated and passed through a column of Bio-Gel P-2. Rel-
evant fractions were freeze dried to give 2 in 79% yield
(16mg, 0.024mmol). MALDI-MS: [M+Na]+ m/z 703.4
expected 703.2. Selected 1H/13C NMR data, D2O,
3.2.2. Methyl 3-O-benzyl-4,6-O-benzylidene-b-D-gluco-
pyranosyl-(1!3)-4,6-O-benzylidene-a-D-glucopyranoside
(6). Disaccharide 5 (1.80g, 2.34mmol) was dissolved in
200mL of CH2Cl2/MeOH 2/1 and 1M NaOMe in
MeOH was added until pH was >12. After 2h, MAL-
DI-MS showed that the reaction was complete. The
reaction mixture was neutralized with Dowex-(H+), fil-
tered and evaporated to dryness. The crude product
70ꢁC, JH-1,
in brackets: B: 5.08 [7.9] (H-1)/100.4
H-2
(C-1), 3.62 (H-2), 3.75 (H-3); A: 5.01 [3.5] (H-1)/100.0
(C-1), 3.99 (H-2), 4.10 (H-3); A0: 4.836 [7.6] (H-1)/
103.6 (C-1), 3.38 (H-2), 3.54 (H-3); B0: 4.832 [7.9]
(H-1)/104.3 (C-1), 3.42 (H-2), 3.58 (H-3). Additional
13C data: 82.4 (C-2B), 80.0 (C-2A), 79.1 (C-3A), 77.0–
68.7 (13 · C-2–C-5), 62.0–61.6 (4 · C-6), 55.7 (OMe).
was recrystallized from CH2Cl2/toluene giving
6
(1.44g, 2.31mmol) in 99%. MALDI-MS: [M+Na]+ m/z
645.6, expected 645.2. 13C NMR: 55.6 (OMe), 63.1–
82.1 (10 · C-2–C-6, 1 · OCH2Ph), 99.9, 101.3, 101.5,
105.4 (4 · acetal C), 126.2–138.4 (aromatic C).
3.2.5. Methyl [2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!2)]-4,6-di-O-acetyl-3-O-benzyl-b-D-glucopyranosyl-
(1!3)-[2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-(1!2)]-
4,6-di-O-acetyl-a-D-glucopyranoside (9). To
a flask
containing tetrasaccharide 8 (118mg, 0.066mmol),
3mL of TFA (90%) was added. The flask was immedi-
ately connected to a rotoevaporator and the solution
evaporated. TLC and MALDI-MS confirmed the com-
plete removal of the two benzylidene groups. The resi-
due was dissolved in CH2Cl2 and washed with aq
NaHCO3 and with water. The organic phase was dried,
filtered and subsequently evaporated. The residue was
dissolved in 2mL of dry MeOH and the pH was ad-
justed to >12 with 1M NaOMe in MeOH. When TLC
and MALDI-MS indicated the complete removal of
the benzoyl groups the solution was neutralized with
Dowex-(H+), filtered and evaporated. To the dry resi-
due, 6mL of pyridine/CH2Cl2 3/1 was added. The solu-
tion was cooled to 0ꢁC with an ice bath and an excess of
3.2.3. Methyl [2,3,4,6-tetra-O-benzoyl-b-D-glucopyrano-
syl-(1!2)]-3-O-benzyl-4,6-O-benzylidene-b-D-glucopyrano-
syl-(1!3)-[2,3,4,6-tetra-O-benzoyl-b-D-glucopyranosyl-
(1!2)]-4,6-O-benzylidene-a-D-glucopyranoside
(8).
Acceptor 6 (100mg, 0.160mmol) and 2,3,4,6-tetra-O-
benzoyl-b-D-glucopyranosyl bromide (317mg,
7
0.480mmol) were dissolved in 8mL of CH2Cl2 and
˚
pre-dried with crushed 4A molecular sieves under argon
at ꢀ55ꢁC for 30min. sym-Collidine (57mg, 0.464mmol)
and AgOTf (164mg, 0.640mmol) were then added.
After 2h, an additional amount of donor (159mg,
0.242mmol) and AgOTf (90mg, 0.350mmol) was added
and the temperature was allowed to rise to 0ꢁC. After
3h, the reaction was quenched by the addition of 3mL

12
E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
Ac2O (1mL) was then added to the stirred solution. The
reaction was monitored by MALDI-MS and TLC. After
20h the reaction was complete and the reaction mixture
was poured onto a slurry of ice and water. When the ice
melted, the phases were separated and the organic phase
was subsequently washed with 1M HCl, aq NaHCO3
and water and finally dried, filtered and evaporated.
The crude product was purified by silica column chro-
matography (gradient, T!T/E: 1/1) to give 9 in 73%
yield (61mg, 0.048mmol). MALDI-MS; [M+Na]+ m/z
1298.8, expected 1297.4. 13C NMR: 20.7–22.0
(12 · Me), 55.3 (OMe), 61.8–73.6 (17 · C-2–C-6), 76.1
(OCH2Ph), 81.4, 81.9, 82.4 (3 · ring C), 99.1, 99.9,
101.2, 101.5 (4 · C-1), 127.5–138.3 (aromatic C),
169.6–171.3 (12 · C@O).
74.6 (17 · C-2–C-5), 78.8, 79.1, 80.5 (3 · ring C),
99.3, 99.4, 99.6, 99.7, 100.8 (5 · C-1), 169.1–170.9
(16 · C@O).
3.2.8. Methyl b-D-glucopyranosyl-(1!3)-[b-D-glucopyr-
anosyl-(1!2)]-b-D-glucopyranosyl-(1!3)-[b-D-glucopyr-
anosyl-(1!2)]-a-D-glucopyranoside (1). Pentasacchar-
ide 12, (22mg, 0.015mmol) was dissolved in 2mL of
MeOH. NaOMe (1M) was added until pH > 12. After
2h MALDI-MS showed that the reaction was complete.
The solution was neutralized with Dowex-(H+), filtered
and subsequently evaporated to dryness. The product
was dissolved in H2O and passed through a column of
Bio-Gel P-2, freeze dried and purified on a column of
Bio-Gel P-2 (2.6 · 68cm) irrigated with water contain-
ing 1% n-butanol to give the target compound 1
(6.8mg, 0.0081mmol) after lyophilization in 54% yield.
MALDI-MS: [M+Na]+ m/z 865.5, expected 865.3.
3.2.6. Methyl [2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!2)]-4,6-di-O-acetyl-b-D-glucopyranosyl-(1!3)-[2,3,4,
6-tetra-O-acetyl-b-D-glucopyranosyl-(1!2)]-4,6-di-O-acet-
yl-a-D-glucopyranoside (10). To a solution of 9 (60mg,
0.047mmol) in 3mL of THF, a catalytic amount of 5%
Pd/C was added. The reaction vessel was then put in a
Parr apparatus and subjected to 100psi of H2 pressure
for 30h when MALDI-MS confirmed the complete for-
mation of the O-debenzylated substance. The reaction
mixture was filtered through a pad of Celite, concen-
trated and purified on silica column chromatography
(gradient, T!T/E: 1/1) to give 10 (53mg, 0.045mmol)
in 95% yield. MALDI-MS: [M+Na]+ m/z 1207.0, ex-
pected 1207.4. 13C NMR: 20.7–21.8 (12 · Me), 55.2
(OMe), 61.7–73.6 (18 · C-2–C-6), 82.0, 84.4 (2 · ring
C), 98.9, 99.4, 101.2, 102.4 (4 · C-1), 169.6–171.3
(12 · C@O).
Selected 1H/13C NMR data, D2O, 70ꢁC, JH-1,
in
H-2
brackets: B: 5.14 [7.8] (H-1)/100.2 (C-1), 3.78 (H-2),
3.91 (H-3); A: 4.98 [3.7] (H-1)/99.9 (C-1), 4.00 (H-2),
4.08 (H-3); B0: 4.88 [8.0] (H-1)/103.6 (C-1), 3.33 (H-2),
3.51 (H-3); C: 4.78 [7.7] (H-1)/103.0 (C-1), 3.38 (H-2),
3.53 (H-3); A0: 4.77 [7.7] (H-1)/103.7 (C-1), 3.34 (H-2),
3.51 (H-3). Additional 13C data: 85.6 (C-3B), 80.6
(C-2B), 80.1 (C-2A), 78.5 (C-3A), 77.1–68.8 (16 · C-2–
C-5), 62.0–61.6 (5 · C-6), 55.6 (OMe).
Acknowledgements
This work was supported by a grant from the Swedish
Research Council.
3.2.7. Methyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-
(1!3)-[2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-(1!2)]-
4,6-di-O-acetyl-b-D-glucopyranosyl-(1!3)-[2,3,4,6-tetra-
O-acetyl-b-D-glucopyranosyl-(1!2)]-4,6-di-O-acetyl-a-
D-glucopyranoside (12). A solution of ethyl 2,3,4,6-tet-
References
1. Salyers, A. A.; Whitt, D. D. Bacterial Pathogenesis: A
Molecular Approach; ASM: Washington, DC, 1994.
2. Jansen, W. T. M.; Snippe, H. Indian J. Med. Res. 2004,
119(Suppl), 7–12.
3. Jansen, W. T. M.; Hogenboom, S.; Thijssen, M. J. L.;
Kamerling, J. P.; Vliegenthart, J. F. G.; Verhoef, J.;
Snippe, H.; Verheul, A. F. M. Infect. Immun. 2001, 69,
787–793.
4. Benaissa-Trouw, B.; Lefeber, D. J.; Kamerling, J. P.;
Vliegenthart, J. F. G.; Kraaijeveld, K.; Snippe, H. Infect.
Immun. 2001, 69, 4698–4701.
5. Adeyeye, A.; Jansson, P.-E.; Lindberg, B.; Henrichsen, J.
Carbohydr. Res. 1988, 180, 295–299.
´ ´
6. Llull, D.; Garcıa, E.; Lopez, R. J. Biol. Chem. 2001, 276,
21053–21061.
7. Nitz, M.; Ling, C.-C.; Otter, A.; Cutler, J. E.; Bundle, D.
R. J. Biol. Chem. 2002, 277, 3440–3446.
8. Adeyeye, A.; Jansson, P.-E.; Kenne, L.; Widmalm, G. J.
Chem. Soc., Perkin Trans. 2 1991, 963–973.
9. Jansson, P.-E.; Kjellberg, A.; Rundlo¨f, T.; Widmalm, G.
J. Chem. Soc., Perkin Trans. 2 1996, 33–37.
10. Garegg, P. J.; Olsson, L.; Oscarson, S. J. Org. Chem. 1995,
60, 2200–2204.
ra-O-acetyl-1-thio-b-D-glucopyranoside
11
(26mg,
0.066mmol) and acceptor 10 (31mg, 0.026mmol) in
˚
1.5mL dry CH2Cl2 was pre-dried with 4A molecular
sieves under argon for 30min. NIS (18mg, 0.080mmol)
and TfOH (0.2lL) was added to initiate the reaction.
After 40min, TLC and MALDI-MS indicated that
product formation had ceased. The reaction was then
quenched by the addition of one drop of Et3N, filtered
through a pad of silica, which was thoroughly washed
with EtOAc. The solution was transferred to a separa-
ting funnel and washed with aq Na2S2O3 and water
and subsequently dried and evaporated. The crude
product was purified by column chromatography (T/E:
3/1!1/3) to give pentasaccharide 12 (23mg,
0.015mmol) in 58% yield. MALDI-MS: [M+Na]+ m/z
1538.6, expected 1537.5. 13C NMR: 20.7–21.6
(16 · Me), 55.6 (OMe), 62.0–62.8 (5 · C-6), 66.9–

E. A. Larsson et al. / Carbohydrate Research 340 (2005) 7–13
13
11. David, S.; Thieffry, A. J. Chem. Soc., Perkin Trans. 1 1979,
1568–1573.
15. Hanessian, S.; Banoub, J. Methods Carbohydr. Chem.
1980, 8, 247–250.
12. Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H.
Tetrahedron Lett. 1980, 31, 1331–1334.
16. Adinolfi, M.; Barone, G.; Guariniello, L.; Iadonisi, A.
Tetrahedron Lett. 1999, 40, 8439–8441.
13. Calinaud, P.; Gelas, J. In Preparative Carbohydrate
Chemistry; Hanessian, S., Ed.; Marcel Dekker: New York,
1997; pp 3–33.
14. Andersson, F.; Fugedi, P.; Garegg, P. J.; Nashed, M.
¨
Tetrahedron Lett. 1986, 27, 3919–3922.
17. Ferrier, R. J.; Furneaux, R. H. Carbohydr. Res. 1976, 52,
63–68.
18. Nordmark, E.-L.; Yang, Z.; Huttunen, E.; Widmalm, G.
Biomacromolecules, 2005, 6, in press.