Block synthesis of Streptococcus pneumoniae type 14 capsular polysaccharide structures

Article abstract of DOI:10.1081/CAR-200066935

Synthesis of a thioglycoside tetrasaccharide block, β-D-Galp-(1 → 4)-β-DGlcp-(1 → 6)-[β-D-Galp-(1 → 4)]-β-D-GlcNPhthp-(1 → SEt, corresponding to the repeating unit of Streptococcus pneumoniae serotype 14 CPS is described. Coupling of this block with a spa

Full text of DOI:10.1081/CAR-200066935

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Block Synthesis of Streptococcus
pneumoniae Type 14 Capsular
Polysaccharide Structures
Andreas Sundgren a , Martina Lahmann a & Stefan Oscarson b
a Department of Chemistry , Göteborg University , Gothenburg,
Sweden
b Department of Organic Chemistry , Arrhenius Laboratory,
Stockholm University , Stockholm, Sweden
Published online: 16 Aug 2006.
To cite this article: Andreas Sundgren , Martina Lahmann & Stefan Oscarson (2005) Block Synthesis
of Streptococcus pneumoniae Type 14 Capsular Polysaccharide Structures , Journal of Carbohydrate
Chemistry, 24:4-6, 379-391
To link to this article: http://dx.doi.org/10.1081/CAR-200066935
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Journal of Carbohydrate Chemistry, 24:379–391, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200066935
Block Synthesis of
Streptococcus pneumoniae
Type 14 Capsular
Polysaccharide Structures
Andreas Sundgren and Martina Lahmann
¨
Department of Chemistry, Goteborg University, Gothenburg, Sweden
Stefan Oscarson
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
Stockholm, Sweden
Synthesis of a thioglycoside tetrasaccharide block, b-D-Galp-(1 ! 4)-b-DGlcp-(1 ! 6)-
[b-D-Galp-(1 ! 4)]-b-D-GlcNPhthp-(1 ! SEt, corresponding to the repeating unit of
Streptococcus pneumoniae serotype 14 CPS is described. Coupling of this block with a
spacer followed by removal of an isopropylidene acetal yielded an acceptor, which was
elongated with the donor block to give a protected dimer of the repeating unit. Iteration
of this methodology yielded the trimer. Deprotection then produced an octa and a dode-
casaccharide derivative ready for conjugation to proteins to afford immunoactive
glycoconjugates.
Keywords Oligosaccharide synthesis, Block synthesis, Glycoconjugate vaccines
INTRODUCTION
The bacterium Streptococcus pneumoniae is a major human pathogen.[1] The
bacterium is divided into serotypes, each serotype corresponding to a unique
structure of the capsular polysaccharide (CPS) surrounding the bacteria.[2]
Received February 9, 2005; accepted March 14, 2005.
In memory of Professor Jacques H. van Boom.
Address correspondence to Stefan Oscarson, Department of Organic Chemistry,
Arrhenius Laboratory, Stockholm University, S-106 91, Stockholm, Sweden.
Fax: þ46 8 15 49 08; E-mail: s.oscarson@organ.su.se
379

A. Sundgren, M. Lahmann, and S. Oscarson
380
Figure 1: Repeating units of S. pneumoniae type 14 CPS.
Serotype 14 is one of the most abundant serotypes with a tetrasaccharide
repeating unit corresponding to a branched Lacto-N-tetraose structure
(Fig. 1).[3]
Both polysaccharide and glycoconjugate vaccines against S. pneumoniae
are now commercial. The polysaccharide vaccine comprises 23 serotypes,
whereas the conjugate vaccine contains seven serotypes, both including
serotype 14. 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 protection.[4–6] To further investi-
gate this, Vliegenthart and coworkers have synthesised type 14 structures up
to octasaccharides and investigated the immunology of some of their protein
conjugates.[7–9] Also, in Jennings’ syntheses of Streptococcus Type III Group B
structures (which is a 3-O-sialylated variant, !6)-[a-NeuAcp-(2 ! 3)-b-D-
Galp-(1 ! 4)]-b-D-GlcNAcp-(1 ! 3)-b-D-Galp-(1 ! 4)-b-D-Glcp-(1!, of type
14,[10]), up to dimeric structures (octasaccharide) of the type 14 CPS have been
prepared.[11] In addition, this group has made a thorough interpretation of the
NMR spectra of both type 14 and Type III Group B oligo- and polysaccharides.[12]
In the ‘80s Kochetkov and coworkers performed polycondensation of a tritylated
tetrasaccharide cyanoorthoester block (b-D-Galp-(1 ! 4)-b-D-GlcNAcp-(1 ! 3)-
b-D-Galp-(1 ! 4)-b-D-Glcp-) to obtain mixtures of polysaccharides with an
average degree of polymerization of about 10.[13,14]
We now report the synthesis of a tetrasaccharide block donor comprising
the type 14 repeating unit and its efficient use in the synthesis of spacer-
containing dimer (octasaccharide) and trimer (dodecasaccharide) derivatives,
to be used in conjugate formation and immunological studies. The synthetic
approach allows further elongation either with a new tetrasaccharide block
or smaller fragments to obtain derivatives with different nonreducing end
groups.
RESULTS AND DISCUSSION
Our strategy involves the use of the branched tetrasaccharide b-D-Galp-
(1 ! 4)-b-D-Glcp-(1 ! 6)-[b-D-Galp-(1 ! 4)]-b-D-GlcNPhthp-(1 ! SEt (9) as
the target building block. Regioselective silylation of compound 1[15] (!2)
followed by regioselective galactosylation afforded disaccharide acceptor 4,
which after desilylation (!5) was regioselectively glycosylated at the
primary position with donor bromide 8, using silver triflate promotion, to
yield the desired tetrasaccharide thioglycoside building block 9 in high yield

S. Pneumoniae type 14 Capsular Polysaccharide Structures
381
Scheme 1: (i) TBDMSCl, pyridine, 08C; (ii) AgOTf, CH2Cl2, 2558C; (iii) Et3N (HF)3, CH2Cl2;
(iv) BzCl, pyridine, 08C; (v) Br2, CH2Cl2, 08C; (vi) AgOTf, CH2Cl2, 2408C; (vii) HO(CH2)3N3, NIS,
AgOTf, CH2Cl2, 2258C; (viii) TFA (90% aqueous).
(61% from 1 over four steps, Sch. 1). Bromide 8 was obtained from the corre-
sponding thioglycoside 7. One advantage of thioglycosides is their easy trans-
formation into other type of donors under mild conditions. Thus, even
instable bromo sugars, for example, containing acid-labile groups, can be syn-
thesized. When derivative 7 was treated with bromine in CH2Cl2, the bromo
sugar was efficiently formed and the acetal was not cleaved off. However, one
of the methyl groups of the isopropylidene acetal was concomitantly monobro-
minated to give a mixture of diastereomers (ratio ꢀ3 : 1). There is precedence in

A. Sundgren, M. Lahmann, and S. Oscarson
382
the literature of similar reactions,[16] and since it did not interfere with the syn-
thetic plans the strategy was not altered.
With the desired building block in hand, the assembly of oligomers
could start. First, a spacer-containing acceptor was synthesized by coupling
3-azidopropanol[17] to compound 9 using NIS/AgOTf as promotor to yield
spacer glycoside 10 (87%), from which the bromoisopropylidene group was
removed by acid hydrolysis to afford the 3,3IV,4IV-triol acceptor 11 (84%,
Sch. 1). NIS-promoted couplings (together with a catalytic amount of either
TfOH or AgOTf) between 11 and thioglycoside donor block 9 were attempted,
but the regioselectivity was low and a mixture of octasaccharides was
obtained. This problem was solved by transforming the thioglycoside 9 into
the corresponding bromide and use of this donor in a silver triflate-promoted
glycosylation.[18] Excellent regioselectivity was observed and the desired
octasaccharide 12 was obtained in 81% yield (Sch. 2). The formed (1 ! 3)-
linkage was proven by NMR. Proton H-4IV could be easily identified by its
shift and coupling constant, thus revealing the H-3IV (3.52 ppm) from a
COSY experiment. H-1V was found to have a significantly stronger NOE to
the H-3IV as compared to H-4IV. Further proof for the identity of all synthesized
derivatives was obtained from the excellent agreement of the 1H NMR data of
the deprotected target structures with published data of the native polysac-
charide and other synthesized type 14 oligosaccharides.[7,8,12]
The synthetic procedure was iterated to yield the trimer. A minor problem
was encountered in the removal of the isopropylidene group, which did not go to
completion. Hence, the reaction was worked up after 4 hr to afford the dimer
acceptor 13 (59%, 88% calculated on consumed starting material) together
with starting material 12 (33%), which were easily separated on a silica gel
column. Formation of a trimer using the same coupling conditions, that is,
the bromo sugar of donor 9 and silver triflate promotion, afforded the dodeca-
saccharide 14 in 77% yield (Sch. 2). Once more, acidic removal of the isopropy-
lidene acetal was sluggish, yielding 59% of 15 (83% calculated on consumed
starting material), which again included the possibility of elongation with
different donors and the formation of larger structures.
Deprotection of the dimer 13 and the trimer 15 was performed in three
´
steps (Sch. 2). Debenzoylation under Zemplen conditions was followed by
removal of the phthalimido groups using ethylenediamine in EtOH. Finally,
N-acetylation with acetic anhydride in MeOH afforded the azido-spacer deriva-
tives in 81% and 73% yield, respectively. The obtained azido derivatives can be
used directly in conjugations with propargyl-activated proteins.[19] To be able to
use other conjugation methodologies, reduction of the azido groups was accom-
plished through catalytic hydrogenolysis to yield the amino derivatives 16 and
17, respectively.
In conclusion, an effective block synthesis of S. pneumoniae type 14 CPS
structures has been worked out. Gram quantities of the building blocks and

Scheme 2: (i) a. Br2, CH2Cl2, 08C; b. AgOTf, CH2Cl2, 2408C; (ii) TFA (90% aqueous); (iii) a. 9 Br2, CH2Cl2, 08C; b. AgOTf, CH2Cl2, 2408C; (iv) NaOMe, MeOH;
(v) Et2N, EtOH, reflux; (vi) Ac2O, MeOH; (vii) H2, Pd/C, HCl, MeOH.

A. Sundgren, M. Lahmann, and S. Oscarson
384
the protected dimers and trimers have been synthesized; further, their high-
yielding transformation into target structures ready for conjugation to
proteins is reported.
EXPERIMENTAL
General Methods
Organic solutions were dried over MgSO4 or Na2SO4 before concentration,
which was performed under reduced pressure at ,458C (bath temperature).
NMR spectra were recorded at 258C and 400 MHz (1H) in CDCl3 with Me4Si
as internal standard (d ¼ 0.00) or 75 or 100 (13C) MHz in CDCl3 with
CDCl3 as internal standard (d ¼ 77.16). TLC was performed on silica gel 60
F254 with detection by charring with 8% sulfuric acid or ninhydrin. Silica
gel (0.040–0.063 mm) was used for column chromatography.
Ethyl (2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-2-deoxy-
2-phthalimido-1-thio-b-D-glucopyranoside (5). Pyridine (9mL) and
TBDMS-Cl (9.35 g, 57.0 mmol) were added to a solution of ethyl 2-deoxy-2-phtha-
limido-1-thio-b-D-glucopyranoside[15] (1, 18.27 g, 51.8 mmol) in CH2Cl2 (140 mL)
at 08C. The reaction was stirred at rt over night. MeOH (30 mL) was added and
the mixture was concentrated and purified by silica gel column chromatography
(toluene-EtOAc 2 : 1) to give ethyl 6-O-tert-butyldimethylsilyl-2-deoxy-2-phthali-
mido-1-thio-b-D-glucopyranoside (2, 28.16 g, 47.7 mmol, 92%). [a]D þ 7.78 (c 0.9,
CHCl3). 13C NMR (CDCl3): d –5.2, –5.2 (SiCH3), 15.1 (SCH2CH3), 18.4
(C(CH3)3), 24.1 (SCH2CH3), 26.1 (C(CH3)3), 55.5, 64.9, 72.7, 74.6, 78.4 (C-2-6),
81.1 (C-1), 123.5–134.3 (aromatic C). Compound 2 (4.22 g, 7.14 mmol) and
2,3,4,6-tetra-O-benzoyl-a-D-galactopyranosyl bromide (3, 4.75 g, 7.24 mmol)
were dried together under vacuum and then dissolved in CH2Cl2 (90 mL)
˚
under an argon atmosphere. 4 A molecular sieves were added and the reaction
mixture was cooled to –558C. After stirring for 25 min, AgOTf (2.80 g,
10.9 mmol) was added and the reaction mixture was stirred for another 45 min
before triethylamine (5.5 mL) was added. After filtration through Celite and con-
centration of the filtrate, the residue was purified by silica gel column chromato-
graphy (toluene - EtOAc 5 : 1) to give ethyl (2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1 ! 4)-6-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimido-1-
thio-b-D-glucopyranoside (4, 6.60 g, 5.64 mmol, 79%). [a]D þ 798 (c 0.5, CHCl3).
13C NMR (CDCl3): d –5.3. –5.1 (SiCH3), 14.9 (SCH2CH3), 18.3 (C(CH3)3), 23.4
(SCH2CH3), 25.9 (C(CH3)3), 55.4, 61.6, 62.5, 68.1, 69.7, 70.6, 71.8, 72.4, 78.7,
80.5 (C-2I-6I, 2II-6II), 81.6 (C-1I), 101.9 (C-1II), 123.3–137.9 (aromatic C),
165.1–166.2 (C55O, Bz), 167.8, 168.3 (C55O, Phth).
Compound 4 (6.60 g, 5.64 mmol) was dissolved in CH2Cl2 (60 mL) and
.
Et3 N (HF)3 (1.0 mL) was added. After stirring for 6 d, the reaction mixture

S. Pneumoniae type 14 Capsular Polysaccharide Structures
385
was diluted with toluene, washed with water, dried, concentrated, and purified
by silica gel chromatography (toluene-EtOAc 2 : 1) to give 5 (4.98 g, 5.35 mmol,
95%). 13C NMR (CDCl3): d 15.0, 24.4 (SCH2CH3), 55.2, 60.9, 62.8, 68.2, 69.7,
70.7, 71.7, 72.5, 78.2, 81.5 (C-2I-6I, 2II-6II), 82.2 (C-1I), 102.3 (C-1II), 123.4–
134.2 (aromatic C), 165.3–166.2 (C55O, Bz), 167.9, 168.4 (C55O, Phth).
[2,6-Di-O-benzoyl-3,4-O-(1-bromomethylethylidene)-b-D-galacto-
pyranosyl]-(1 ! 4)-2,3,6-tri-O-benzoyl-a-D-glucopyranosyl bromide
(8). Benzoyl chloride (45 mL) was added to a solution of ethyl (3,4-O-isopropy-
lidene-b-D-galactopyranosyl)-(1!4)-1-thio-b-D-glucopyranoside[20] (6, 22.30 g,
52.7 mmol) in pyridine (110 mL) at 08C. After stirring for 40 min, ice (50 mL)
and toluene (200 mL) were added and the mixture was stirred for another
60 min. The organic phase was washed with water and NaHCO3 solution
(sat.), dried, concentrated, and purified by silica gel chromatography
(toluene-EtOAc 6 : 1) to produce ethyl [2,6-di-O-benzoyl-3,4-O-(1-methylethyli-
dene)-b-D-galactopyranosyl]-(1 ! 4)-2,3,6-tri-O-benzoyl-1-thio-b-D-glucopyra-
noside (7, 47.86 g, 50.6 mmol, 96 %). [a]D þ 398 (c 1.1, CHCl3). 13C NMR (CDCl3):
d 15.3, 24.8 (SCH2CH3), 26.6, 27.8 (C(CH3)2), 63.2, 63.3, 71.1, 71.7, 73.6, 74.1,
74.2, 75.7, 77.5, 77.8 (C-2I-6I, 2II-6II), 84.1 (C-1I), 100.5 (C-1II), 111.3 (C(CH3)2),
128.5–133.8 (aromatic C), 165.3–166.4 (C55O, Bz). Compound 7 (8.56 g,
9.04 mmol) was dissolved in CH2Cl2 (55 mL) and bromine (1.7 mL, 33 mmol)
was added at 08C. The reaction was stirred for 80 min and then quenched by
the addition of cyclohexene (1.5 mL) and triethylamine (2 mL). The solution
was concentrated, coevaporated with toluene, and purified by silica gel
column chromatography (pentane-toluene 3:1 to 1 : 10) to give 8 (7.42 g,
7.12 mmol, 79%). 13C NMR (CDCl3):
d
24.5 (C(CH3)(CH2Br)), 37.2
(C(CH3)(CH2Br)), 61.8, 62.7, 70.3, 71.2, 71.6, 73.2, 73.5, 74.5, 74.6, 78.0 (C-2I-
6I, 2II-6II), 86.9 (C-1I), 100.5 (C -1II), 109.4 (C(CH3)(CH2Br)), 128.4–133.8
(aromatic C), 165.0–166.0 (C55O, Bz).
Ethyl [2,6-di-O-benzoyl-3,4-O-(1-bromomethylethylidene)-b-D-gala-
ctopyranosyl]-(1 ! 4)-(2,3,6-tri-O-benzoyl-b-D-glucopyranosyl)-(1!6)-
[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)]-2-deoxy-2-
phthalimido-1-thio-b-D-glucopyranoside (9). A solution of 5 (3.12 g,
˚
3.35 mmol) and 8 (3.36 g, 3.22 mmol) in CH2Cl2 (25 mL) containing 4 A molecu-
lar sieves was stirred for 30 min. AgOTf (1.05 g, 4.11 mmol) was added and
the mixture stirred for 1 hr at –408C. Triethylamine was added and the
mixture filtered through Celite, concentrated, and dried under reduced pressure.
The residue was purified by silica gel column chromatography (toluene-EtOAc
5 : 1) to give 9 (5.36 g, 2.83 mmol, 88%). 13C NMR (CDCl3): d 14.6, 23.6
(SCH2CH3), 24.4 (C(CH3)(CH2Br)), 36.9 (C(CH3)(CH2Br)), 54.7, 62.3, 62.5, 62.6,
67.3, 67.8, 69.9, 70.8, 71.1, 71.1, 71.6, 71.9, 72.4, 72.5, 73.3, 74.6, 75.2, 76.5,
77.9, 81.0 (C-2I-IV-6I-IV), 82.6, 100.4, 100.8, 101.6 (C-1I-IV), 109.4
(C(CH3)(CH2Br)), 123.0–137.7 (aromatic C), 164.6–165.9 (C55O, Bz and Phth).

A. Sundgren, M. Lahmann, and S. Oscarson
386
3-Azidopropyl
(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-
(2,3,6-tri-O-benzoyl-b-D-glucopyranosyl)-(1!6)-[(2,3,4,6-tetra-O-benzoyl-
b-D-galactopyranosyl)-(1 ! 4)]-2-deoxy-2-phthalimido-b-D-glucopyrano-
side (11). A solution of 9 (742 mg, 392 mmol) and 3-azidopropanol[17] (104 mg,
˚
1.03 mmol) in CH2Cl2 (10 mL) containing 4 A molecular sieves was stirred for
30 min. The mixture was then cooled to –358C, NIS (140 mg, 622 mmol) and a
catalytic amount of AgOTf were added, and stirring was continued for another
25 min at 2258C. Triethylamine was added and the mixture filtered through
Celite and concentrated. The residue was purified by silica gel column chromato-
graphy (toluene-EtOAc 3 : 1) to yield 3-azidopropyl [2,6-di-O-benzoyl-3,4-O-(1-
bromomethylethylidene)-b-D-galactopyranosyl]-(1!4)-(2,3,6-tri-O-benzoyl-b-D-
glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-(1!4)]-
2-deoxy-2-phthalimido-b-D-glucopyranoside (10, 659 mg, 341mmol, 87%). 13C
NMR (CDCl3):
d 24.6 (C(CH3)(CH2Br)), 28.7 (OCH2CH2CH2N3), 37.0
(C(CH3)(CH2Br)), 48.0 (OCH2CH2CH2N3), 55.6, 62.3, 62.6, 66.1, 67.2, 67.8,
69.8, 70.0, 71.1, 71.3, 71.6, 71.9, 72.4, 72.6, 72.6, 73.4, 74.7, 75.3, 78.0, 82.7
(C-2I-IV-6I-IV, OCH2CH2CH2N3), 98.5, 100.5, 100.9, 101.7 (C -1I-IV), 109.5
(C(CH3)(CH2Br)), 125.3–137.9 (aromatic C), 164.6–166.0 (C55O, Bz and
Phth). Compound 10 (1.603 g, 829 mmol) was dissolved in 90% TFA (20 mL),
stirred for 40 min, concentrated, and purified by silica gel column chromato-
graphy (toluene-EtOAc 3 : 1) to give 11 (1.263 g, 696 mmol, 84%). [a]D þ578
(c 0.7, CHCl3). 13C NMR (CDCl3):
d
28.8 (OCH2CH2CH2N3), 48.1
(OCH2CH2CH2N3), 55.7, 62.0, 62.5, 62.6, 66.2, 67.2, 67.9, 68.6, 69.9, 70.1,
71.2, 71.7, 72.0, 72.4, 72.7, 72.7, 72.8, 72.8, 73.8, 75.8, 82.7 (C-2I-IV-6I-IV
,
OCH2CH2CH2N3), 98.6, 100.9, 101.1, 101.8 (C-1I-IV), 123.4–138.0 (aromatic
C), 164.8–166.4 (C55O Bz, Phth).
3-Azidopropyl (2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1!4)-(2,3,6-
tri-Obenzoyl-b-D-glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1 ! 4)]-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-
(1!3)-(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1!4)-(2,3,6-tri-O-benzoyl-
b-D-glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyra-
nosyl)-(1!4)]-2-deoxy-2-phthalimido-b-D-glucopyranoside (13). Bromine
(0.20 mL, 4.0 mmol) was added to a solution of 9 (1.061 g, 561 mmol) in CH2Cl2
(10 mL) at 08C. After stirring for 90 min at 08C, the reaction mixture was
concentrated, coevaporated with toluene, and dried under vacuum. The
˚
residue was redissolved in CH2Cl2 (20 mL) containing 4 A molecular sieves
together with 11 (910 mg, 501 mmol), and the mixture was stirred for 30 min
at rt. The mixture was cooled to 2408C and AgOTf (285 mg, 1.117 mmol) was
added. Triethylamine was added after 25 min and the mixture was filtered
through Celite and concentrated. The residue was purified by silica gel
column chromatography (toluene -EtOAc 6 : 1) to produce 3-azidopropyl [2,6-
di-O-benzoyl-3,4-O-(1-bromomethylethylidene)-b-D-galactopyranosyl]-(1!4)-

S. Pneumoniae type 14 Capsular Polysaccharide Structures
387
(2,3,6-tri-O-benzoyl-b-D-glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-
D-galactopyranosyl)-(1!4)]-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-(1!3)-
(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-(2,3,6-tri-O-benzoyl-b-D-gluco-
pyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)]-2-
deoxy-2-phthalimido-b-D-glucopyranoside (12, 1.480 g, 406 mmol, 81%). 13C
NMR (CDCl3):
d 24.7 (C(CH3)(CH2Br)), 28.8 (OCH2CH2CH2N3), 37.1
(C(CH3)(CH2Br)), 48.1 (OCH2CH2CH2N3), 53.6, 55.2, 55.6, 61.7, 62.3, 62.4,
62.6, 64.1, 66.1, 67.1, 67.9, 67.9, 68.8, 69.4, 69.7, 69.8, 70.0, 70.8, 70.9, 71.1,
71.2, 71.6, 71.9, 72.1, 72.2, 72.6, 72.8, 72.9, 73.2, 73.4, 74.6, 75.1, 75.7, 78.0,
80.9, 82.6, 83.3 (C-2I-VIII-6I-VIII, OCH2CH2CH2N3), 98.5, 98.6, 100.8, 100.8,
101.6, 101.7, 101.8 (C-1I-VIII), 109.5 (C(CH3)(CH2Br)),128.4–134.9 (aromatic
C), 164.0–166.2 (C55O, Bz and Phth). A solution of compound 12 (1.202 g,
330 mmol) in TFA (25 mL, 85% aqueous) was stirred for 3.5 hr, then concen-
trated, coevaporated with toluene, and purified by silica gel column chromato-
graphy (toluene-EtOAc 4 : 1) to give 13 (687 mg, 195 mmol, 59%) and unreacted
12 (397mg, 109mmol, 33%). [a]D þ 628 (c 1.1, CHCl3). 13C NMR (CDCl3): d 28.8
(OCH2CH2CH2N3), 48.1 (OCH2CH2CH2N3), 55.1, 55.6, 61.7, 62.5, 62.7,
64.3, 66.1, 67.1, 67.9, 68.0, 68.5, 69.2, 69.5, 69.7, 69.9, 70.0, 70.8, 71.1, 71.2,
71.6, 72.0, 72.2, 72.2, 72.4, 72.6, 72.8, 73.0, 73.1, 73.2, 73.7, 75.1, 76.0,
80.9, 82.6, 83.6 (C-2I-VIII-6I-VIII
, OCH2CH2CH2N3), 98.5, 100.8, 101.4,
101.7, 101.8, 101.8 (C -1I-VIII), 128.3–137.9 (aromatic C), 164.0–166.3 (C55O, Bz
and Phth). Anal. calcd. for C193H165N5O61: C, 65.66; H, 4.71; N, 1.98. Found: C,
65.53; H, 4.74; N, 1.86.
3-Azidopropyl [2,6-di-O-benzoyl-b-D-galactopyranosyl]-(1!4)-(2,3,6-
tri-O-benzoyl-b-D-glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1!4)]-(2-deoxy-2-phthalimido-b-D-glucopyranosyl)-
(1 ! 3)-(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-(2,3,6-tri-O-
benzoyl-b-D-glucopyranosyl)-(1!6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galac-
topyranosyl)-(1!4)]-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-(1!3)-
(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1!4)-(2,3,6-tri-O-benzoyl-b-D-
glucopyranosyl)-(1!6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!4)]-2-deoxy-2-phthalimido-b-D-glucopyranoside
(15).
Bromine
(0.10 mL, 2.0 mmol) was added to a solution of compound 9 (303 mg, 160mmol)
in CH2Cl2 (5mL) and the reaction mixture was stirred at 08C for 90 min, then
concentrated, coevaporated with toluene, and dried under vacuum. The
residue was dissolved in CH2Cl2 (8mL) followed by the addition of 13 (450 mg,
˚
128 mmol) and 4 A molecular sieves and stirred for 50min at rt. AgOTf (85 mg,
333 mmol) was added and the mixture was stirred for 30min at 2408C. Triethyl-
amine was added and the mixture was filtered through Celite and concentrated.
The residue was purified by silica gel chromatography (toluene-EtOAc 6 : 1) to
produce 3-azidopropyl [2,6-di-O-benzoyl-3,4-O-(1-bromomethylethylidene)-b-D-
galactopyranosyl]-(1 ! 4)-(2,3,6-tri-O-benzoyl-b-D-glucopyranosyl)-(1 ! 6)-[(2,

A. Sundgren, M. Lahmann, and S. Oscarson
388
3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)]-(2-deoxy-2-phthalimido-
b-D-glucopyranosyl)-(1 ! 3)-(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-
(2,3, 6-tri-O-benzoyl-b-D-glucopyranosyl)-(1 ! 6)-[(2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1 ! 4)]-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-
(1 ! 3)-(2,6-di-O-benzoyl-b-D-galactopyranosyl)-(1 ! 4)-(2,3,6-tri-O-benzoyl-
b-D-glucopyranosyl)-(1!6)-[(2,3,4,6-tetra-O-benzoyl-b-D-galactopyranosyl)-
(1!4)]-2-deoxy-2-phthalimido-b-D-glucopyranoside (14, 531mg, 99mmol, 77%).
13C NMR (CDCl3): d 24.7 (C(CH3)(CH2Br)), 28.8 (OCH2CH2CH2N3), 37.1
(C(CH3)(CH2Br)), 48.1 (OCH2CH2CH2N3), 55.2, 55.6, 62.4, 62.7, 64.0, 66.1,
67.1, 67.7, 67.8, 67.9, 68.0, 69.4, 69.5, 69.5, 69.6, 69.6, 69.7, 69.9, 70.0, 70.3,
70.9, 71.1, 71.2, 71.3, 71.4, 71.6, 71.7, 71.8, 71.8, 71.9, 72.1, 72.2, 72.3, 72.4,
72.4, 72.5, 72.6, 72.7, 72.8, 72.9, 73.0, 73.1, 73.2, 73.4, 73.4, 78.1, 80.6, 81.0,
81.1, 82.5, 82.5, 82.6, 82.7, 82.9, 83.1 (C-2I-XII-6I-XII, OCH2CH2CH2N3), 100.8,
101.0, 101.1, 101.1, 101.2, 101.3, 101.4, 101.5, 101.7, 101.7, 102.0 (C-1I-XII),
128.0–135.0 (aromatic C), 164.0–168.2 (C55O, Bz and Phth). Compound 14
(490 mg, 91 mmol) was dissolved in TFA (20 mL, 85% aqueous). The mixture
was stirred for 4 hr, then evaporated, coevaporated with toluene, and purified
by silica gel column chromatography (toluene-EtOAc 4 : 1) to give 15 (281 mg,
54 mmol, 59%) and unreacted 14 (142 mg, 26 mmol, 29%). [a]D þ 728 (c 3,
CHCl3). 13C NMR (CDCl3): d 28.8 (OCH2CH2CH2N3), 48.1 (OCH2CH2CH2N3),
55.2, 55.6, 61.5, 61.7, 62.0, 62.3, 62.6, 63.9, 63.9, 66.1, 67.0, 67.6, 67.8,
67.8, 67.9, 68.0, 68.3, 68.9, 69.4, 69.6, 69.7, 69.7, 69.8, 70.0, 70.8, 71.1, 71.2,
71.6, 71.7, 71.9, 72.1, 72.2, 72.4, 72.4, 72.6, 72.7, 72.8, 72.9, 72.9, 73.0,
73.2, 75.0, 75.2, 75.8, 77.7, 80.6, 81.0, 82.6, 83.4, (C-2I-XII-6I-XII
,
OCH2CH2CH2N3), 98.5, 98.9, 100.8, 101.0, 101.2, 101.3, 101.6, 101.7, 101.7,
101.8 (C-1I-XII), 128.3–135.0 (aromatic C), 164.0–168.2 (C55O, Bz and Phth).
Anal. calcd. for C288H244N6O91: C, 65.95; H, 4.69; N, 1.60. Found: C, 65.78; H,
4.60; N, 1.48.
3-Aminopropyl (b-D-galactopyranosyl)-(1!4)-(b-D-glucopyranosyl)-
(1!6)-[(b-D-galactopyranosyl)-(1!4)]-(2-deoxy-2-acetamido-b-D-gluco-
pyranosyl)-(1!3)-(b-D-galactopyranosyl)-(1!4)-(b-D-glucopyranosyl)-
(1!6)-[(b-D-galactopyranosyl)-(1!4)]-2-deoxy-2-acetamido-b-D-gluco-
pyranoside (16). Derivative 13 (36 mg, 10 mmol) was dissolved in MeOH (6 mL)
and 1 M NaOMe (100 mL in MeOH) was added. The solution was stirred over-
night, neutralized with dry ice, and concentrated. The residue was passed
through a reversed phase column (H2O), concentrated, dissolved in EtOH
(10 mL) and ethylenediamine (1 mL), and refluxed for 4 d. The reaction mixture
was concentrated, and the formed crude product filtered through a reversed
phase column (H2O) and then purified on a Bio -Gel P-2 column (1% 1-butanol
in H2O). The combined carbohydrate -containing fractions were concentrated
and the residue was dissolved in MeOH (5 mL) and acetic anhydride (50 mL)
was added. After stirring for 10 min the solution was concentrated, coevaporated

S. Pneumoniae type 14 Capsular Polysaccharide Structures
389
with MeOH and toluene, purified on a Bio-Gel P-2 column (1% 1-butanol in
H2O), and concentrated. Pd/C (catalytic amount) and 1 M HCl (40 mL) were
added to the product from the last step dissolved in MeOH (1.5 mL), stirred
for 15 min under H2 (1 atm.), filtered through a reversed phase column, con-
centrated, coevaporated with water, and dried under vacuum to yield
16 (11 mg, 7.6 mmol, 76%). [a]D –1.78 (c 0.8, H2O). 1H NMR (D2O) (selected
data): d 1.93 (t, 2H, CH2 3-aminopropyl), 2.02, 2.03 (2s, 6H, NHAc),
3.07 (t, 2H), 3.35 (t, 2H), 4.15 (d, 1H, H-4IV), 4.27 (t, 2H, H-6I, 6V), 4.43
(t, 2H, H-1IV, 1VIII), 4.51–4.55 (m, 5H, H-1I, 1II, 1III, 1VI, 1VII), 4.69 (d, 1H,
H-1V). MALDI-TOF MS: Calcd. for C55H95N3O41 ([M þ Na]þ): 1476.5; found
1477.8.
3-aminopropyl (b-D-galactopyranosyl)-(1!4)-(b-D-glucopyranosyl)-
(1!6)-[(b-D-galactopyranosyl)-(1!4)]-(2-deoxy-2-acetamido-b-D-gluco-
pyranosyl)-(1!3)-(b-D-galactopyranosyl)-(1!4)-(b-D-glucopyranosyl)-
(1!6)-[(b-D-galactopyranosyl)-(1!4)]-(2-deoxy-2-acetamido-b-D-gluco-
pyranosyl)-(1!3)-(b-D-galactopyranosyl)-(1!4)-(b-D-glucopyranosyl)-
(1!6)-[(b-D-galactopyranosyl)-(1!4)]-2-deoxy-2-acetamido-b-D-gluco-
pyranoside (17). Compound 15 (31 mg, 6 mmol) was deprotected as described
for compound 13 above to give 17 (7 mg, 3.3 mmol, 55 %). [a]D–2.18 (c 0.3, H2O).
1H NMR (D2O) (selected data): d 1.97 (t, 2H, CH2 3-aminopropyl), 2.02, 2.02,
2.03 (3s, 9H, NHAc), 4.15 (2d, 2H, H-4IV, 4VIII), 4.26 (2t, 3H, H-6I, 6V, 6IX),
4.43 (2t, 3H, H-1IV, 1VIII, 1XII), 4.51–4.55 (m, 7H, H-1I, 1II, 1III, 1VI, 1VII, 1X,
1XI), 4.69 (2d, 2H, H-1V, 1IX). MALDI-TOF MS: Calcd. for C81H138N4O61
([M þ Na]þ): 2165.8; found 2168.3.
ACKNOWLEDGMENTS
¨
We are indebted to Professor Goran Karlsson at the Swedish NMR Centre for
help with the interpretation of the NMR spectra of the octasaccharide 13.
Financial support from the Swedish Research Council is gratefully
acknowledged.
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