Synthesis of Streptococcus pneumoniae type 3 neoglycoproteins varying in oligosaccharide chain length, loading and carrier protein

Article abstract of DOI:10.1002/1521-3765(20011015)7:20<4411::AID-CHEM4411>3.0.CO;2-T

The preparation is described of a range of neoglycoproteins containing synthesised fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3, that is β-D-GlcpA(1 → 4)-β-D-Glcp-(1 → O)-(CH₂)₃NH₂ (1), β-D-Glcp-(1 → 3)-β-D-GlcpA-(1 → 4)β-D-Glcp-(1 → O)-(CH₂)₃NH₂ 2, and β-D-GlcpA-(1 → 4)-β-D-Glcp-(1 → 3)-βD-GlcpA-(1 → 4)-β-D-Glcp-(1 → O)(CH₂)₃NH₂ (3). A blockwise approach was developed for the synthesis of the protected carbohydrate chains, in which the carboxylic groups were introduced prior to deprotection by selective oxidation of HO-6 in the presence of HO-4 by using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy radical). After deprotection, the 3-aminopropyl spacer of the fragments was elongated with diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dione) and the elongated oligosaccharides were conjugated to CRM₁₉₇ (cross-reacting material of diphtheria toxin), KLH (keyhole limpet hemocyanin) or TT (tetanus toxoid). The resulting neoglycoconjugates varied in oligosaccharide chain length, oligosaccharide loading and protein carrier. These well-defined conjugates are ideal probes for evaluating the influence of the different structural parameters in immunological tests.

Full text of DOI:10.1002/1521-3765(20011015)7:20<4411::AID-CHEM4411>3.0.CO;2-T

FULL PAPER
Synthesis of Streptococcus pneumoniae Type 3 Neoglycoproteins Varying
in Oligosaccharide Chain Length, Loading and Carrier Protein
Dirk J. Lefeber, Johannis P. Kamerling, and Johannes F. G. Vliegenthart*[a]
Abstract: The preparation is described
of a range of neoglycoproteins contain-
ing synthesised fragments of the capsu-
lar polysaccharide of Streptococcus
pneumoniae type 3, that is b-d-GlcpA-
(1 ! 4)-b-d-Glcp-(1 ! O)-(CH2)3NH2
(1), b-d-Glcp-(1 ! 3)-b-d-GlcpA-(1 ! 4)-
b-d-Glcp-(1 ! O)-(CH2)3NH2 (2), and
b-d-GlcpA-(1 ! 4)-b-d-Glcp-(1 ! 3)-b-
d-GlcpA-(1 ! 4)-b-d-Glcp-(1 ! O)-
(CH2)3NH2 (3). A blockwise approach
was developed for the synthesis of the
protected carbohydrate chains, in which
the carboxylic groups were introduced
prior to deprotection by selective oxida-
tion of HO-6 in the presence of HO-4 by
using TEMPO (2,2,6,6-tetramethyl-1-pi-
peridinyloxy radical). After deprotec-
tion, the 3-aminopropyl spacer of the
fragments was elongated with diethyl
squarate (3,4-diethoxy-3-cyclobutene-
1,2-dione) and the elongated oligosac-
charides were conjugated to CRM197
(cross-reacting material of diphtheria
toxin), KLH (keyhole limpet hemocya-
nin) or TT (tetanus toxoid). The result-
ing neoglycoconjugates varied in oligo-
saccharide chain length, oligosaccharide
loading and protein carrier. These well-
defined conjugates are ideal probes for
evaluating the influence of the different
structural parameters in immunological
tests.
Keywords: antigens ´ neoglycocon-
jugate vaccines ´ oligosaccharides ´
oxidation ´ protecting groups
type C,[6] and Streptococcus pneumoniae serotypes.[7] These
neoglycoproteins are prepared by conjugation of isolated
capsular polysaccharides or a mixture of polysaccharide-
derived oligosaccharides to a protein carrier. The polysac-
charide ± protein conjugates have a very complex and unde-
fined structure, whereas the oligosaccharide ± protein conju-
gates contain the carbohydrate as mixtures with respect to
chain length and presented epitope. Especially in the case of a
pneumococcal conjugate vaccine, wherein many serotypes
have to be included, the presence of mixtures complicates the
analysis of the products, which is increasingly important for
product control. The use of synthetic polysaccharide-related
oligosaccharide fragments with a unique structure could help
to solve these problems. Furthermore, it creates the oppor-
tunity for the preparation of tailor-made neoglycoproteins
that can be used for vaccination, for ELISA tests not hindered
by bacterial contamination,[8] and for studying the immune
response at a molecular level. Although synthetic neo-
glycopeptides contain a defined carbohydrate and a defined
peptide part, the use of neoglycoproteins with a defined
carbohydrate part allows a comparison with the currently
used vaccines.
Introduction
The Gram positive bacterium Streptococcus pneumoniae is
one of the most prevalent infectious pathogens, which causes
life-threatening diseases such as meningitis, pneumonia and
otitis media. In the past, these infections could be efficiently
treated with antibiotics. However, death rates have not
declined and furthermore, resistance to pneumococcal strains
is still growing.[1] Immunocompetent people can be protected
efficiently by vaccination with the available 23-valent capsular
polysaccharide (CPS) vaccines.[2] The highest incidence of
pneumococcal infections is, however, in young children, the
elderly and immunocompromised patients. People in these
groups do not benefit from the mentioned vaccines, since they
do not respond adequately to the T-cell independent poly-
saccharides.[3] However, conjugation of carbohydrate antigens
to a protein results in a T-cell dependent neoglycoconjugate
antigen that can give an efficient immune response in the
population at high risk.[4]
Currently, neoglycoconjugate vaccines are in use against
Haemophilus influenzae type b,[5] Neisseria meningitidis
Recent studies with Streptococcus pneumoniae type 6B
neoglycoproteins showed that a synthetic tetrasaccharide
fragment, that is one repeating unit of the 6B CPS, coupled to
KLH was sufficient to generate a protective antibody
response in mice.[9] An overview of the synthetic oligosac-
charides from several serotypes of S. pneumoniae has been
[a] Prof. Dr. J. F. G. Vliegenthart, D. J. Lefeber, Prof. Dr. J. P. Kamerling
Bijvoet Center, Department of Bio-Organic Chemistry
Utrecht University
P.O. Box 80.075, 3508 TB Utrecht (The Netherlands)
Fax : (‡31) 30 2540980
E-mail: j.f.g.vliegenthart@chem.uu.nl
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D. J. Lefeber et al.
FULL PAPER
given by Kamerling,[10] and an overview of well-defined
neoglycoconjugates related to several encapsulated bacteria
by Pozsgay.[11]
To obtain well-defined products for the simplification of
product control, and to investigate the influence of oligosac-
charide chain length, oligosaccharide loading and carrier
protein on the immunogenicity of the neoglycoproteins, here
we present the development of a versatile synthetic route for
the preparation of oligosaccharides related to the CPS of S.
pneumoniae type 3 and their conjugation to a protein in
varying ratios. For the conjugation, the defined coupling
chemistry of diethyl squarate
Figure 1. Synthesised oligosaccharide fragments to be coupled to a carrier
protein.
(3,4-diethoxy-3-cyclobutene-
1,2-dione) was applied, and as
carrier proteins were used
CRM197 (cross-reacting materi-
al of diphtheria toxin) and TT
(tetanus toxoid), which are rel-
evant for human vaccination,
and KLH (keyhole limpet he-
mocyanin), which is suitable for
animal studies.
Results and Discussion
Retrosynthetic strategy: The
CPS of S. pneumoniae type 3
consists of b(1 ! 3)-linked cel-
lobiuronic acid repeating units.
A route for the synthesis of
Scheme 1. Retrosynthetic strategy.
small fragments has been reported by Chernyak et al.,[12]
based on 3,6-lactonisation of glucuronic acid containing
structures to prepare acceptors with a free HO-3 group for
the introduction of the (1 ! 3)-linkage. However, the yields
for lactonisation and opening of the lactone were moderate in
the synthesis of the disaccharide acceptor and can be expected
to be even lower in the preparation of larger fragments.
During the progress of our work, the synthesis of a protected
disaccharide was described by Garegg et al.[13] In contrast to
these earlier reports, in our approach the carboxylic functions
are introduced in a late stage of the synthetic route by
regioselective oxidation using TEMPO. This approach avoids
elaborate protecting group manipulations and possibly low
yielding coupling steps. Furthermore, the blockwise strategy
employing disaccharide building blocks paves the way for the
versatile preparation of larger fragments.
benzylidene protecting groups. This selective oxidation elim-
inates the need for protection of HO-4. After debenzoylation,
the azide can be hydrogenated and the resulting aminopropyl
spacer coupled via diethyl squarate to lysine residues of
CRM197, KLH or TT. The tetrasaccharide acceptor can be
elongated to obtain longer fragments by coupling with the
mentioned disaccharide or other donors.
Synthesis of the saccharide fragments: Monosaccharide
acceptor allyl 2,3,6-tri-O-benzoyl-b-d-glucopyranoside (7)
was prepared as depicted in Scheme 2. Benzoylation of allyl
4,6-O-benzylidene-b-d-glucopyranoside (4)[14] by using ben-
zoyl chloride (!5, 92%), and subsequent debenzylidenation
with trifluoroacetic acid afforded 6 in a yield of 92%.
Selective benzoylation of the primary hydroxyl function with
benzoyl imidazole[15] gave monosaccharide acceptor 7 in 95%
yield.
For the synthesis of imidate donor 12 (Scheme 2), selective
protection of allyl 4,6-O-benzylidene-b-d-glucopyranoside (4)
was attempted. However, several methods, for example, using
stannylene acetals (Bu2SnO),[16] benzoyl chloride at 508C,
benzoyl imidazole, heterogeneous catalysis with tetrabutyl-
ammonium iodide and potassium carbonate,[17] or silyla-
tion,[18] failed to give satisfactory isolated yields of compounds
with a protecting group on HO-2 or HO-3. In the related a-
glucoside of 4 the acidity of HO-2 is increased by H-bond
formation with the anomeric oxygen,[19] thereby increasing the
difference in reactivity between HO-2 and HO-3. For this
For the synthesis of the target oligosaccharide fragments 1,
2 and 3 (Figure 1), three monosaccharide building blocks were
designed (see Scheme 1).
Coupling of imidate donor 12 with acceptor 7 or 17 results
in the formation of disaccharide 19 or 22, respectively.
Dechloroacetylation of 22 results in an acceptor for the
synthesis of the tri- and tetrasaccharide fragments. Disacchar-
ide 19 can be deallylated and imidated to obtain a disacchar-
ide donor, which can be coupled with 22 to give 30.
Introduction of the carboxylic groups is achieved by selective
2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) mediated ox-
idation of the primary hydroxyl groups after liberation of the
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Oligosaccharides
4411±4421
The preparation of disaccharide donor 21 is presented in
Scheme 4. Coupling of donor 12 with acceptor 7 using 5%
TMSOTf as a promoter afforded disaccharide 19 in an
excellent yield of 96%. Deallylation with tris(triphenylphos-
phine)rhodium(i) chloride and N-iodosuccinimide (!20,
70%) and subsequent trichloroacetimidation gave disacchar-
ide donor 21 (88%).
Scheme 2. Synthesis of the monosaccharide building blocks 7 and 12.
a) PhCOCl, C5H5N, CH2Cl2, 92%; b) CF3COOH, H2O, CH2Cl2, 92%;
c) PhCOCl, imidazole, CH2Cl2, 95%; d) PhCOCl, imidazole, CH2Cl2,
74%; e) ClAc-Cl, C5H5N, CH2Cl2, 96%; f) i) [(PPh3)3RhICl], CH3C6H5/
EtOH 9:4, ii) NIS, H2O, THF, 77%; g) Cl3CCN, DBU, CH2Cl2, 93%.
Scheme 4. Synthesis of disaccharide donor 21. a) 5% TMSOTf, CH2Cl2,
96%; b) i) [(PPh3)3RhICl], DABCO, CH2Cl2/CH3C6H5/EtOH 0.6:9:4,
ii) NIS, H2O, THF, 70%; c) Cl3CCN, DBU, CH2Cl2, 88%.
In Scheme 5, the strategies for the preparation of the
disaccharide acceptor 23 and target disaccharide 1 are shown.
Test-oxidations with TEMPO[25] of monosaccharide 6 showed
that the allyl group was incompatible with the reaction
conditions. Therefore, the 3-azidopropyl spacer was intro-
duced prior to oxidation. Coupling of disaccharide imidate 21
to 3-azido-1-propanol 18 to afford 22 was very troublesome
and gave irreproducible yields of around 50% due to
orthoester formation (J1,2 ˆ 5.1 Hz). Coupling via the glycosyl
bromide, prepared from 20 by reaction with Vilsmeier
reagent,[26] gave similar problems. Alternatively, the synthesis
of disaccharide 22 was achieved by coupling of donor 12 and
the spacer-containing monosaccharide acceptor 17 using 8%
TMSOTf as a promoter in a yield of 75%. Subsequent
dechloroacetylation with diazabicyclo[2.2.2]octane (DAB-
CO)[27] gave disaccharide acceptor 23 (98%).
reason, selective benzoylation of allyl 4,6-O-benzylidene-a-d-
glucopyranoside (8)[20] was performed with benzoyl imidazole,
which resulted in the formation of 9 in a good yield of 74%.
Subsequent chloroacetylation[21] (!10, 96%), deallylation by
isomerisation of the double bond with Wilkinsonꢁs catalyst[22]
and removal of the 1-propenyl group with N-iodosuccinimide
and water[23] (!11, 77%), and trichloroacetimidation[24]
afforded monosaccharide donor 12 (93%).
Monosaccharide acceptor 17, containing a 3-azidopropyl
spacer, was prepared from 5 (Scheme 3). Deallylation of 5 by
For the synthesis of the target disaccharide fragment 1, first
23 was debenzylidenated using trifluoroacetic acid (!24,
90%). Then, selective oxidation by using TEMPO and
aqueous sodium hypochlorite afforded the protected cello-
Scheme 3. Synthesis of the monosaccharide building block 17.
a) i) [(PPh3)3RhICl], CH3C6H5/EtOH 5:2, ii) NIS, H2O, THF, 75%;
b) Cl3CCN, DBU, CH2Cl2, 82%; c) 8% TMSOTf, CH2Cl2, 65%;
d) CF3COOH, H2O, CH2Cl2, 63%; e) PhCOCl, imidazole, CH2Cl2, 96%.
1
biuronic acid derivative 25 (85%). H NMR analysis after
methylation by using diazomethane proved the identity of the
compound (d ˆ 3.37, COOCH3). Debenzoylation of 25 using
sodium methoxide in methanol at pH 11, followed by hydro-
genation of the azide should give 1. However, using the
using Wilkinsonꢁs catalyst and N-iodosuccinimide (!13,
75%) and subsequent trichloroacetimidation afforded mono-
saccharide donor 14 (82%).
Coupling of 14 with 3-azido-1-
propanol (18), prepared in 74%
from 3-bromo-1-propanol by
treatment with sodium azide,
was performed in dichlorome-
thane using 8% TMSOTf as a
promoter (!15, 65%). Deben-
zylidenation of 15 by using tri-
fluoroacetic acid (!16, 63%)
and selective benzoylation of
Scheme 5. Synthesis of disaccharide acceptor 23 and target disaccharide 1. a) 8% TMSOTf, CH2Cl2, 78%;
b) 15 equiv DABCO, CH3C6H5/EtOH 1:1, 558C, 98%; c) CF3COOH, H2O, CH2Cl2, 90%; d) TEMPO, aqueous
NaOCl, KBr, Bu4NBr, aqueous NaCl, aqueous NaHCO3, CH2Cl2, 85%; e) i) NaOMe, MeOH (pH 11),
ii) NaBH4, 10% Pd/C, H2O, 90%.
HO-6 with benzoyl imidazole
afforded acceptor 17 in 96%
yield.
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D. J. Lefeber et al.
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conventional hydrogenolysis method with 10% Pd/C and
hydrogen, a side product in about 20% was generated.
1H NMR analysis showed the removal of some of the spacer
signals, but the full identity of the compound could not be
disclosed. When using dithiothreitol and DBU,[28] the same
side-product was formed. Reduction with sodium borohy-
dride and 10% Pd/C in H2O afforded disaccharide fragment 1
in 90% yield over two steps with the formation of only small
amounts of the mentioned side-product.
Focusing on target trisaccharide fragment 2, the synthesis of
protected trisaccharide 27 (Scheme 6) was quite troublesome.
Scheme 7. Synthesis of target tetrasaccharide 3. a) 10% TMSOTf, CH2Cl2,
75%; b) thiourea, EtOH/C5H5N 8:1, 908C, 84%; c) CF3COOH, H2O,
CH2Cl2, 70%; d) TEMPO, aqueous NaOCl, KBr, Bu4NBr, aqueous NaCl,
aqueous NaHCO3, CH2Cl2, 65%; e) i) NaOMe, MeOH (pH 11), ii)
NaBH4, 10% Pd/C, H2O, 60%.
tion using TEMPO and aqueous sodium hypochlorite gave 33
1
in a yield of 65%. H NMR analysis after methylation with
diazomethane showed two singlets at d ˆ 3.39 and 3.34
(COOCH3). Deprotection of 33 as described for 25 gave
tetrasaccharide fragment 3 in 60% yield over two steps.
In order to make a comparison possible of the immuno-
logical studies of the aimed di-, tri- and tetrasaccharide ±
protein conjugates with relevant blanks, the glucose and
glucuronic acid conjugates were also prepared. To this end,
3-aminopropyl glucopyranoside (35) and 3-aminopropyl glu-
copyranosiduronic acid (37) (Scheme 8) were prepared from
16. Debenzoylation of 16 (!34), followed by reduction of the
Scheme 6. Synthesis of target trisaccharide 2. a) NIS, TfOH, Et2O/CH2Cl2
1:2, 82%; b) CF3COOH, H2O, CH2Cl2, 70%; c) TEMPO, aqueous NaOCl,
KBr, Bu4NBr, aqueous NaCl, aqueous NaHCO3, CH2Cl2, 76%; d) i)
NaOMe, MeOH (pH 11), ii) NaBH4, 10% Pd/C, H2O, 89%.
Coupling of 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl tri-
chloroacetimidate with 23 using 10% TMSOTf as a promoter
gave a complex reaction mixture. The use of the correspond-
ing glucosyl bromide or thioglucoside as a donor resulted in
the same complex mixture with one of the main side-products
being the orthoester (J1'',2'' ˆ 5.2 Hz). Coupling of 2,3,4,6-tetra-
O-benzoyl-a-d-glucopyranosyl trichloroacetimidate with 23
using 10% TMSOTf as a promoter gave trisaccharide 27 in
70% yield. The yield was, however, irreproducible due to the
formation of orthoester and some other side-products. Finally,
the use of ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-b-d-glucopy-
ranoside (26) as donor was found to result in an efficient
glycosylation of 23. Activation with triflic acid and N-
iodosuccinimide gave trisaccharide 27 in a reproducible yield
of 82%. Debenzylidenation using trifluoroacetic acid (!28,
70%) and subsequent oxidation of HO-6 using TEMPO and
aqueous sodium hypochlorite gave the protected trisaccharide
fragment 29 (76%) (1H NMR: d ˆ 3.62, d, J4',5' ˆ 9.3 Hz, 1H;
H-5'). Deprotection of 29 as described for 25 afforded
trisaccharide fragment 2 in 89% yield over two steps.
Scheme 8. Synthesis of 3-aminopropyl glucopyranoside 35 and 3-amino-
propyl glucopyranosiduronic acid 37. a) NaOMe, MeOH (pH 10);
b) NaBH4, 10% Pd/C, H2O, 88%; c) TEMPO, aqueous NaOCl, KBr,
H2O; d) NaBH4, 10% Pd/C, H2O, 80%.
azide with sodium borohydride and 10% Pd/C in water gave
35 in 88% over two steps. Oxidation of 34 with TEMPO and
aqueous sodium hypochlorite in water gave 36, which was
purified by conventional acetylation with acetic anhydride
and pyridine, column chromatography, and deacetylation
using sodium methoxide. Glucuronic acid derivative 37 was
obtained after reduction of 36 as described for 34 (80% over
three steps).
The 1H NMR data of the target saccharide fragments 1 ± 3,
35 and 37, derived from two-dimensional TOCSY and
ROESY measurements, are presented in Table 1.
In the synthesis of target tetrasaccharide fragment 3
(Scheme 7), as a first step disaccharide donor 21 was coupled
with disaccharide acceptor 23 using 10% TMSOTf as a
promoter, resulting in 30 (75%). Dechloroacetylation of 30
using thiourea (!31, 84%), followed by debenzylidenation
using trifluoroacetic acid (!32, 70%), and selective oxida-
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Oligosaccharides
4411±4421
Table 1. 500 MHz 1H NMR data of 1, 2, 3, 35, and 37 at 300 K (in ppm).[a]
J1,2 couplings are presented in parentheses.
H-1
H-2
3.29
H-3
3.50
H-4
3.38
H-5
3.48
H-6a
3.93
H-6b
3.73
35
37
1
4.49
(7.9)
4.48
(7.9)
4.51
(7.7)
4.51
(7.7)
4.51
(7.7)
4.54
(8.3)
4.79
(7.7)
4.51
(8.2)
4.54
(7.7)
4.82
(8.2)
4.51
(8.2)
3.33
3.33
3.36
3.33
3.57
3.36
3.33
3.58
3.38
3.36
3.51
3.66
3.52
3.66
3.79
3.51
3.65
3.79
3.68
3.52
3.51
3.61
3.52
3.61
n.d.[b]
3.40
3.62
n.d.
3.72
3.61
3.75
3.61
n.d.
3.46
n.d.
n.d.
n.d.
3.76
3.98
3.98
3.81
3.80
1'
2
Figure 2. Coupling of saccharide fragments to
a
protein via diethyl
2'
2''
3
squarate. a) EtOH/sodium phosphate buffer (0.1m, pH 6.9); b) sodium
borate buffer (0.1m, pH 9.5); CRM197, KLH or TT.
3.92
3.98
3.72
3.81
Table 2. Oligosaccharide loading and coupling efficiency for the conjuga-
tion of 1, 2, 3, 35 and 37 to CRM197
.
3'
3''
3'''
Entry
Glycoconjugate
Oligosaccharide
loading [molmol
Coupling
efficiency [%]
3.61
3.52
3.98
3.82
1
]
1
2
3
4
5
6
7
8
9
CRM197 ± 35
CRM197 ± 37
CRM197 ± 1
CRM197 ± 1
CRM197 ± 2
CRM197 ± 2
CRM197 ± 2
CRM197 ± 3
CRM197 ± 3
CRM197 ± 3
8.5
6.6
3.1
6.7
4.9
6.8
12.0
2.9
6.7
8.1
77
67
56
52
45
41
54
26
32
37
[a] The signals for the 3-aminopropyl spacer are similar for all compounds:
d ˆ 4.03 and 3.84 (OCH2CH2CH2NH2), 3.15 (OCH2CH2CH2NH2), 2.00
(OCH2CH2CH2NH2). [b] n.d. ˆ not determined.
Preparation of the protein conjugates: Studying neoglyco-
conjugates in which only one parameter is varied is important
to understand the different factors that influence the immu-
nogenicity of the conjugates. To this end, focusing on CPS-
related oligosaccharides from S. pneumoniae type 3, neo-
glycoproteins with a different oligosaccharide chain length,
carbohydrate ± protein ratio, and protein carrier have been
prepared.
10
CRM197-conjugates by MALDI-TOF was possible only when
about 10mm sodium borate buffer was still present in the
sample. Attempts to measure the molecular mass in water
failed.
The saccharide fragments described above were conjugated
to CRM197, TT or KLH, using diethyl squarate as linker
(Figure 2).[29, 30] Elongation of the saccharides with diethyl
squarate was performed in ethanol/0.1m sodium phosphate
(pH 6.9). The reaction products were purified by solid-phase
extraction and coupled to protein in 0.1m sodium borate
buffer at pH 9.5.
CRM197 is a non-toxic product of a single mutation in the
diphtheria toxin gene[31] and exists as a pure and well-defined
protein that is often used as a carrier in human neoglycocon-
jugate vaccines.[32] As can be
To investigate the immunological effect of the protein
carrier, two other proteins were coupled with the saccharide
fragments. Tetanus toxoid (TT), prepared by inactivation of
the crude toxin with formalin, is frequently used in human
immunisation studies, and KLH is frequently used for
immunisation studies in animals. The results for the coupling
of the saccharide fragments to TT and KLH are shown in
Table 3. Since TT and KLH are heterogeneous protein
preparations, the carbohydrate loading of these conjugates
could not be derived reliably by MALDI-TOFanalysis. This is
seen from the coupling efficien-
cies in Table 2, the yield for the
conjugation of the saccharides
to CRM197 decreases when
charged structures are coupled
(entries 1 and 2), or when the
oligosaccharides become larger
(entries 1, 3, 5, and 8). The
amount of carbohydrate incor-
poration (Table 2) was meas-
ured by MALDI-TOF analysis
(see Figure 3 for some repre-
sentative spectra). It should be
noted that analysis of the
Figure 3. MALDI-TOF spectra of CRM197 and entries 4, 6 and 10 (Table 2).
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D. J. Lefeber et al.
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with CH2Cl2, washed twice with 10% (w/v) aqueous NaCl, and the organic
layer was dried, filtered, and concentrated. The residue was purified by
column chromatography (CH2Cl2) to obtain 5 as a white solid (16.2 g,
92%). Rf ˆ 0.81 (CH2Cl2/EtOAc 9:1); [a]2D0 ˆ ‡19 (c ˆ 1); 1H NMR
(CDCl3): d ˆ 7.98 ± 7.94, 7.39 ± 7.29 (2m, 15H; PhCH, 2PhCO), 5.84 ± 5.70
due to their intrinsic heterogeneous nature. Therefore, the
loading was determined by analysis of the carbohydrate
(Dubois)[33] and protein content (Pierce).[34]
ˆ
(m, 1H; OCH2CH CH2), 5.79 (t, J2,3 ˆ 9.5, J3,4 ˆ 9.5 Hz, 1H; H-3), 5.54 (s,
Table 3. Oligosaccharide loading and coupling efficiency for the conjuga-
tion of 1, 2 and 3 to KLH and TT.
1H; PhCH), 5.51 (dd, J1,2 ˆ 7.8 Hz, 1H; H-2), 5.27 ± 5.10 (m, 2H;
ˆ
OCH2CH CH2), 4.85 (d, 1H; H-1), 4.43 (dd, J5,6a ˆ 4.9, J6a,6b ˆ 10.3 Hz,
ˆ
1H; H-6a), 4.39 ± 4.32, 4.17 ± 4.09 (2m, each 1H; OCH2CH CH2), 3.94 (t,
Entry
Glycoconjugate
Oligosaccharide
loading [mgmg
Coupling
efficiency [%]
1
J4,5 ˆ 9.5 Hz, 1H; H-4), 3.88 (t, J5,6b ˆ 10.3 Hz, 1H; H-6b), 3.69 (ddd, 1H;
]
H-5); 13C NMR (CDCl3): d ˆ 165.4, 165.0 (2PhCO), 117.6
1
2
3
4
5
6
KLH ± 1
KLH ± 2
KLH ± 3
TT± 1
TT± 2
TT± 3
14.1
13.3
24.6
31.3
23.6
22.9
9
15
9
16
11
8
ˆ
(OCH2CH CH2), 101.3 (PhCH), 100.3 (C-1), 78.7, 72.3, 72.0, 66.5 (C-
ˆ
2,3,4,5), 70.1 (OCH2CH CH2), 68.5 (C-6); elemental analysis calcd (%) for
C30H28O8 (516.6): C 69.76, H 5.46; found: C 69.88, H 5.38.
Allyl 2,3-di-O-benzoyl-b-d-glucopyranoside (6): CF3COOH (1.64 mL) and
H2O (0.22 mL) were added to a solution of 5 (1.50 g, 2.90 mmol) in CH2Cl2
(40 mL). The mixture was stirred for 3 h, diluted with CH2Cl2, washed with
10% (w/v) aqueous NaHCO3 (2 Â ) and 10% (w/v) aqueous NaCl, and the
organic layer was dried, filtered, and concentrated. The residue was
purified by column chromatography (CH2Cl2/MeOH 98:2) to obtain 6
(1.14 g, 92%). Rf ˆ 0.15 (CH2Cl2/acetone 9:1); [a]2D0 ˆ ‡90 (c ˆ 1); 1H NMR
(CDCl3): d ˆ 7.97 ± 7.94, 7.51 ± 7.26 (2m, 10H; 2PhCO), 5.85 ± 5.72 (m, 1H;
In conclusion, we have synthesised a range of neoglyco-
proteins related to S. pneumoniae type 3. The immunological
properties of these well-defined neoglycoconjugates will be
evaluated, in order to gain insight in the structural factors
influencing the immune response of this type of neoglyco-
conjugate vaccines. Preliminary results of the immunisation of
mice with the CRM197-conjugates show that they can induce a
protective immune response.[35]
ˆ
ˆ
OCH2CH CH2), 5.27 ± 5.10 (m, 2H; OCH2CH CH2), 4.78 (d, J1,2 ˆ 7.7 Hz,
ˆ
1H; H-1), 4.38 ± 4.31, 4.18 ± 4.11 (2m, each 1H; OCH2CH CH2), 3.58 (ddd,
J4,5 ˆ 9.6, J5,6a, J5,6b ˆ 3.6, 4.5 Hz, 1H; H-5); 13C NMR (CDCl3): d ˆ 165.2
ˆ
(2PhCO), 117.6 (OCH2CH CH2), 99.8 (C-1), 77.3, 75.7, 71.3, 69.9 (C-
ˆ
2,3,4,5), 70.1 (OCH2CH CH2), 62.1 (C-6); elemental analysis calcd (%) for
C23H24O8 (428.4): C 64.48, H 5.65; found: C 64.32, H 5.64.
Allyl 2,3,6-tri-O-benzoyl-b-d-glucopyranoside (7): Benzoyl chloride
(0.15 mL, 1.28 mmol) was added dropwise to a solution of imidazole
(0.15 g, 2.2 mmol) in CH2Cl2 (4 mL). The suspension was filtered, and the
filtrate was added dropwise to a solution of 6 (0.50 g, 1.17 mmol) in CH2Cl2
(4 mL). After stirring for 36 h, the mixture was diluted with CH2Cl2,
washed with 10% (w/v) aqueous NaCl, and the organic layer was dried,
filtered, and concentrated. The crude residue was purified by column
chromatography (CH2Cl2/EtOAc 95:5) to obtain 7 (0.59 g, 95%). Rf ˆ 0.73
(CH2Cl2/EtOAc 9:1); [a]D20 ˆ ‡63 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.09 ±
Experimental Section
General: All chemicals were of reagent grade, and were used without any
further purification. A solution of tetanus toxoid (TT; 6.12 mgmL 1) was
obtained from the Dutch National Institute of Health (RIVM, Bilthoven,
The Netherlands); a solution of keyhole limpet hemocyanin (KLH;
ˆ
7.94, 7.47 ± 7.33 (2m, 15H; 3PhCO), 5.85 ± 5.72 (m, 1H; OCH2CH CH2),
1
50 mgmL
) was obtained from the Eijkman ± Winkler Institute for
Microbiology (Utrecht University, Utrecht, The Netherlands); a solution
of a non-toxic variant of diphtheria toxin (cross-reacting material, CRM197
ˆ
5.24 ± 5.07 (m, 2H; OCH2CH CH2), 4.81 (d, J1,2 ˆ 7.6 Hz, 1H; H-1), 4.75
(dd, J5,6b ˆ 4.5, J6a,6b ˆ 12.1 Hz, 1H; H-6b), 4.68 (dd, J5,6a ˆ 2.5 Hz, 1H;
;
ˆ
H-6a), 4.38 ± 4.31, 4.18 ± 4.10 (2m, each 1H; OCH2CH CH2), 3.83 (ddd,
61.15 mgmL 1) was obtained from Chiron (Siena, Italy). Reactions were
monitored by TLC on plastic silica gel 60 F254 (Merck); after examination
under UV light, compounds were visualised by heating with 10% (v/v)
ethanolic H2SO4, orcinol (2 mgmL 1) in 20% (v/v) methanolic H2SO4, or
ninhydrin (1.5 mgmL 1) in BuOH/H2O/HOAc (38:1.75:0.25). In the work-
up procedures of reaction mixtures, organic solutions were washed with
appropriate amounts of the indicated aqueous solutions, then dried
(MgSO4), and concentrated under reduced pressure at 20 ± 408C on a
water-bath. Column chromatography was performed on silica gel 60
(Merck, 0.063 ± 0.200 mm). Optical rotations were measured in CHCl3,
unless stated otherwise, with a Perkin ± Elmer 241 polarimeter, using a
10 cm 1 mL cell. 1H NMR spectra in CDCl3 were recorded at 278C with a
Bruker AC 300 spectrometer; the dH values are given in ppm relative to the
signal for internal Me4Si (d ˆ 0). 13C (APT, 75 MHz) NMR spectra in
CDCl3 were recorded at 278C with a Bruker AC 300 spectrometer;
indicated ppm values for dC are relative to the signal of CDCl3 (d ˆ 76.9).
1H NMR spectra in D2O were recorded at 278C with a Bruker AMX 500
spectrometer, and the dH values are given in ppm relative to the signal for
internal acetone (d ˆ 2.225). Two-dimensional TOCSY and ROESY
spectra in D2O were recorded using a Bruker AMX 500 spectrometer
(500 MHz) at 278C to assign the spectra of compounds 1, 2, 3, 35 and 37.
Fast-atom-bombardment mass spectrometry (FABMS) was performed on a
JEOL JMS SX/SX 102A four-sector mass spectrometer. Matrix-assisted
laser desorption ionisation time-of-flight (MALDI-TOF) spectra were
obtained on a Voyager-DE mass spectrometer using 2,4-dihydroxybenzoic
acid (DHB) in H2O as a matrix. Elemental analyses were carried out by H.
Kolbe Mikroanalytisches Laboratorium (Mülheim an der Ruhr, Germany).
J4,5 ˆ 9.7 Hz, 1H; H-5); 13C NMR (CDCl3): d ˆ 167.1, 166.8, 165.1 (3PhCO),
ˆ
117.5 (OCH2CH CH2), 99.6 (C-1), 76.5, 74.3, 71.4, 69.5 (C-2,3,4,5), 69.9
ˆ
(OCH2CH CH2), 63.4 (C-6); elemental analysis calcd (%) for C30H28O9
(532.5): C 67.66, H 5.29; found: C 67.53, H 5.20.
Allyl 2-O-benzoyl-4,6-O-benzylidene-a-d-glucopyranoside (9): Benzoyl
chloride (0.68 mL, 5.81 mmol) was added dropwise to a solution of
imidazole (0.80 g, 11.73 mmol) in CH2Cl2 (10 mL). The suspension was
filtered and the filtrate was added dropwise to a solution of allyl 4,6-O-
benzylidene-a-d-glucopyranoside (8)[20] (1.50 g, 4.86 mmol) in CH2Cl2
(9.5 mL). After stirring at boiling under reflux for 36 h, the mixture was
diluted with CH2Cl2, washed with 10% (w/v) aqueous NaCl, and the
organic layer was dried, filtered, and concentrated. The crude residue was
purified by column chromatography, whereby the 2,3-di-O-benzoylated
compound was eluted with toluene/EtOAc 95:5, and 9 with toluene/EtOAc
9:1 (1.48 g, 74%). Rf ˆ 0.61 (toluene/EtOAc 7:3); [a]2D0 ˆ ‡99 (c ˆ 1);
1H NMR (CDCl3): d ˆ 8.09 ± 8.06, 7.56 ± 7.35 (2m, 10H; PhCH, PhCO),
ˆ
5.87 ± 5.74 (m, 1H; OCH2CH CH2), 5.54 (s, 1H; PhCH), 5.30 ± 5.22, 5.15 ±
ˆ
5.10 (2m, each 1H; OCH2CH CH2), 5.20 (d, J1,2 ˆ 3.8 Hz, 1H; H-1), 5.04
(dd, J2,3 ˆ 9.6 Hz, 1H; H-2), 4.36 (brt, J3,4 ˆ 9.4 Hz, 1H; H-3), 4.29 (dd,
J6a,6b ˆ 10.2, J5,6a ˆ 4.8 Hz, 1H; H-6a), 3.75 (t, J5,6b ˆ 10.2 Hz, 1H; H-6b),
3.59 (t, 1H; H-4), 2.68 (s, 1H; HO-2); 13C NMR (CDCl3): d ˆ 166.0
(PhCO), 136.9 (PhCH, quaternary C), 129.4 (PhCO, quaternary C), 117.5
ˆ
(OCH2CH CH2), 101.8 (PhCH), 95.8 (C-1), 81.3, 73.8, 68.7, 62.2 (C-2,3,4,5),
ˆ
68.7, 68.6 (C-6, OCH2CH CH2); elemental analysis calcd (%) for C23H24O7
(412.4): C 66.98, H 5.86; found:C 67.18, H 5.76.
Allyl 2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-a-d-glucopyrano-
side (10): Chloroacetyl chloride (70 mL, 0.88 mmol) was added to a cooled
(58C) solution of 9 (0.35 g, 0.84 mmol) in CH2Cl2 (30 mL) and pyridine
(1.5 mL). After stirring for 2 h, another portion of chloroacetyl chloride
(35 mL, 0.44 mmol) was added, and stirring was continued for 1 h. Then, the
Allyl 2,3-di-O-benzoyl-4,6-O-benzylidene-b-d-glucopyranoside (5): Ben-
zoyl chloride (10.3 mL, 88.7 mmol) was added dropwise to a solution of
allyl 4,6-O-benzylidene-b-d-glucopyranoside (4)[14] (10.5 g, 34.1 mmol) in
CH2Cl2 (80 mL) and pyridine (10 mL). After 3 h, the mixture was diluted
4416
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Oligosaccharides
4411±4421
mixture was co-concentrated with toluene, diluted with CH2Cl2, and
washed with 10% (w/v) aqueous NaCl. The organic layer was dried,
filtered, and concentrated. The residue was purified by column chroma-
tography (toluene/EtOAc 95:5) to afford 10 (0.38 g, 96%). Rf ˆ 0.50
(toluene/EtOAc 9:1); [a]D20 ˆ ‡104 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.04 ±
8.02, 7.35 ± 7.48 (2m, 10H; PhCH, PhCO), 5.88 ± 5.75 (m, 1H;
2,3-Di-O-benzoyl-4,6-O-benzylidene-d-glucopyranosyl trichloroacetimi-
date (14): Cl3CCN (4.1 mL) and DBU (0.14 mL) were added at 08C to a
solution of 13 (1.82 g, 3.82 mmol) in dry CH2Cl2 (65 mL). After 3 h, the
mixture was concentrated, and the residue was purified by column
chromatography. Impurities were eluted with toluene/EtOAc 95:5, and
14 was obtained by elution with toluene/EtOAc 9:1 (1.88 g, 82%; a/b ˆ
1
ˆ
OCH2CH CH2), 5.87 (t, J2,3 ˆ 9.9, J3,4 ˆ 9.8 Hz, 1H; H-3), 5.54 (s, 1H;
3:2). Rf ˆ 0.68 (toluene/EtOAc 8:2); H NMR (CDCl3): d ˆ 8.72 (s, 0.4H;
PhCH), 5.31 ± 5.23, 5.16 ± 5.12 (2m, each 1H; OCH2CH CH2), 5.28 (d,
NHb), 8.60 (s, 0.6H; NHa), 6.76 (d, J1,2 ˆ 3.8 Hz, 0.6H; H-1a), 6.23 (d, J1,2
ˆ
ˆ
J1,2 ˆ 3.8 Hz, 1H; H-1), 5.12 (dd, 1H; H-2), 4.33 (dd, J6a,6b ˆ 10.3, J5,6a
ˆ
7.2 Hz, 0.4H; H-1b), 6.17 (t, J2,3 ˆ 9.8, J3,4 ˆ 9.9 Hz, 0.6H; H-3a), 5.84 (dd,
J2,3 ˆ 8.2, J3,4 ˆ 9.1 Hz, 0.4H; H-3b), 5.75 (t, 0.4H; H-2b), 5.60 (s, 0.6H;
PhCHa), 5.57 (s, 0.4H; PhCHb), 5.56 (dd, 0.6H; H-2a); 13C NMR (CDCl3):
d ˆ 165.3 (2PhCO), 160.7 (OC(NH)CCl3), 101.5 (PhCH), 95.5, 93.6 (C-
1a,1b), 68.4 (C-6).
4.8 Hz, 1H; H-6a), 4.00, 3.94 (2 d, Jgem ˆ 14.8 Hz, each 1H; ClCH2CO), 3.81
(t, J5,6b ˆ 10.3 Hz, 1H; H-6b), 3.78 (t, J4,5 ˆ 9.6 Hz, 1H; H-4); 13C NMR
(CDCl3): d ˆ 166.3, 165.7 (PhCO, ClCH2CO), 136.6 (PhCH, quaternary C),
ˆ
128.7 (PhCO, quaternary C), 117.8 (OCH2CH CH2), 101.5 (PhCH), 95.7
ˆ
(C-1), 78.8, 72.0, 70.8, 62.5 (C-2,3,4,5), 68.7, 68.6 (C-6, OCH2CH CH2), 40.4
(ClCH2CO); elemental analysis calcd (%) for C25H25ClO8 (488.9): C 61.41,
3-Azidopropyl 2,3-di-O-benzoyl-4,6-O-benzylidene-b-d-glucopyranoside
(15): A solution of 3-azido-1-propanol 18 (1.4 g, 14 mmol) and 14 (1.80 g,
2.90 mmol) in dry CH2Cl2 (35 mL), containing 4 Š molecular sieves, was
stirred under Ar for 1 h. Then, TMSOTf (42 mL, 0.23 mmol) was
introduced, and after 30 min, the mixture was neutralised with dry
pyridine, filtered over cotton, diluted with CH2Cl2, and washed with 10%
(w/v) aqueous NaCl. The organic layer was dried, filtered, and concen-
trated. The residue was purified by column chromatography (toluene/
EtOAc 95:5) to obtain 15 (1.05 g, 65%). Rf ˆ 0.70 (toluene/EtOAc 8:2);
[a]2D0 ˆ ‡19 (c ˆ 1); 1H NMR (CDCl3): d ˆ 7.98 ± 7.94, 7.52 ± 7.25 (2m, 15H,
PhCH, 2PhCO), 5.78 (t, J2,3 ˆ 9.5, J3,4 ˆ 9.5 Hz, 1H; H-3), 5.55 (s, 1H;
H 5.15; found: C 61.22, H 5.03.
2-O-Benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-d-glucopyranose (11):
Tris(triphenylphosphine)rhodium(i) chloride (70 mg) was added to
a
solution of 10 (0.10 g, 0.21 mmol) in absolute EtOH (20 mL) and toluene
(45 mL). After stirring at boiling under reflux for 2.5 h, TLC (toluene/
EtOAc 9:1) showed the formation of a new spot (Rf ˆ 0.52), and the
mixture was concentrated. The residue was dissolved in THF (18 mL), and
water (2 mL) and NIS (0.13 g) were added. After 20 min, the mixture was
concentrated, diluted with CH2Cl2, washed with 10% (w/v) aqueous
NaHSO3 (2 Â ) and 10% (w/v) aqueous NaCl, and the organic layer was
dried, filtered, and concentrated. The crude residue was purified by column
chromatography. Impurities were eluted with toluene/EtOAc 9:1, and 11
was obtained as a light brown syrup by elution with toluene/EtOAc 8:2
(71 mg, 77%; a/b ˆ 4:1). Rf ˆ 0.29 (toluene/EtOAc 9:1); 1H NMR (CDCl3):
d ˆ 8.04 ± 8.02, 7.46 ± 7.34 (2m, 10H; PhCH, PhCO), 5.54 (s, 0.8H; PhCHa),
5.52 (s, 0.2H; PhCHb), 5.88 (t, J2,3 ˆ 9.9, J3,4 ˆ 9.8 Hz, 1H; H-3), 5.57 (d,
J1,2 ˆ 3.6 Hz, 0.8H; H-1a), 5.19 (dd, J1,2 ˆ 7.9 Hz, 0.2H; H-2b), 5.10 (dd,
0.8H; H-2a), 4.92 (d, 0.2H; H-1b), 4.01, 3.95 (2d, Jgem ˆ 14.8 Hz, each 1H;
ClCH2CO); 13C NMR (CDCl3): d ˆ 166.6, 165.8 (PhCO, ClCH2CO), 136.6
(PhCH, quaternary C), 101.5 (PhCH), 95.8 (C-1a), 90.7 (C-1b), 68.8 (C-6a),
68.3 (C-6b), 40.4 (ClCH2COa), 40.3 (ClCH2COb).
PhCH), 5.46 (dd, J1,2 ˆ 7.8 Hz, 1H; H-2), 4.79 (d, 1H; H-1), 4.44 (dd, J6a,6b
10.3, J5,6a ˆ 4.9 Hz, 1H; H-6a), 3.93 (t, J4,5 ˆ 9.6 Hz, 1H; H-4), 3.88 (t, J5,6b
ˆ
ˆ
10.1 Hz, 1H; H-6b), 3.70 (dt, 1H; H-5), 3.98 (dt, 1H; OCHHCH2CH2N3),
3.61 (ddd, 1H; OCHHCH2CH2N3), 3.29 ± 3.17 (m, 2H; OCH2CH2CH2N3),
1.83 ± 1.72 (m, 2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ 136.6
(PhCH, quaternary C), 101.7, 101.4 (PhCH, C-1), 78.7, 72.4, 71.9, 66.6 (C-
2,3,4,5), 68.5 (C-6), 66.7 (OCH2CH2CH2N3), 47.7 (OCH2CH2CH2N3), 28.9
(OCH2CH2CH2N3); elemental analysis calcd (%) for C30H29O8N3 (559.5): C
64.39, H 5.22; found: C 64.25, H 5.27.
3-Azidopropyl 2,3-di-O-benzoyl-b-d-glucopyranoside (16): CF3COOH
(1.0 mL) and H2O (0.16 mL) were added to a solution of 15 (1.24 g,
2.21 mmol) in CH2Cl2 (40 mL). The mixture was stirred for 3 h, diluted with
CH2Cl2, washed with 10% (w/v) aqueous NaHCO3 (2 Â ) and 10% (w/v)
aqueous NaCl, and the organic layer was dried, filtered, and concentrated.
The residue was purified by column chromatography (CH2Cl2/EtOAc
9:1 ! CH2Cl2/acetone 6:4) to obtain 16 (0.66 g, 63%). Rf ˆ 0.07 (CH2Cl2/
EtOAc 9:1); [a]2D0 ˆ ‡37 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.01 ± 7.94, 7.51 ±
7.29 (2m, 10H, 2PhCO), 4.73 (d, J1,2 ˆ 7.9 Hz, 1H; H-1), 3.59 (ddd, 1H;
OCHHCH2CH2N3), 3.33 ± 3.17 (m, 2H; OCH2CH2CH2N3), 1.83 ± 1.73 (m,
2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ 101.0 (C-1), 77.1, 75.8, 71.3,
2-O-Benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-a-d-glucopyranosyl tri-
chloroacetimidate (12): Cl3CCN (2.7 mL) and DBU (87 mL) were added to
a solution of 11 (1.06 g, 2.43 mmol) in dry CH2Cl2 (28 mL). After 2 h, the
mixture was concentrated, and the residue was purified by column
chromatography (toluene/EtOAc 95:5) to obtain 12 (1.31 g, 93%). Rf ˆ
0.49 (toluene/EtOAc 9:1); [a]2D0 ˆ ‡67 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.60
(s, 1H; NH), 8.01 ± 7.98, 7.48 ± 7.36 (2m, 10H; PhCH, PhCO), 6.71 (d, J1,2
ˆ
3.8 Hz, 1H; H-1), 5.94 (t, J2,3 ˆ 9.9, J3,4 ˆ 9.9 Hz, 1H; H-3), 5.59 (s, 1H;
PhCH), 5.40 (dd, 1H; H-2), 4.41 (dd, J5,6a 4.9, J6a,6b ˆ 10.4 Hz, 1H; H-6a),
4.21 (dt, J4,5 ˆ 9.9, J5,6b ˆ 10.2 Hz, 1H; H-5), 4.03, 3.97 (2d, Jgem ˆ 14.8 Hz,
each 1H; ClCH2CO), 3.92 (t, 1H; H-4), 3.84 (t, 1H; H-6b); 13C NMR
(CDCl3): d ˆ 166.3, 165.3 (PhCO, ClCH2CO), 160.6 (OC(NH)CCl3), 136.3
(PhCH, quaternary C), 101.6 (PhCH), 93.4 (C-1), 78.1, 70.8, 70.5, 65.1 (C-
2,3,4,5), 68.4 (C-6), 40.4 (ClCH2CO); elemental analysis calcd (%) for
C24H21Cl4NO8 (593.1): C 48.59, H 3.56; found: C 48.10, H 3.42.
69.9
(C-2,3,4,5),
66.3
(OCH2CH2CH2N3),
62.1
(C-6),
47.7
(OCH2CH2CH2N3), 28.9 (OCH2CH2CH2N3); elemental analysis calcd
(%) for C23H25O8N3 (471.4): C 58.59, H 5.34; found: C 58.32, H 5.40.
3-Azidopropyl 2,3,6-tri-O-benzoyl-b-d-glucopyranoside (17): Benzoyl
chloride (0.20 mL, 1.69 mmol) was added dropwise to a solution of
imidazole (0.14 g, 2.11 mmol) in CH2Cl2 (10 mL). The suspension was
filtered, and the filtrate was added dropwise to a solution of 16 (0.66 g,
1.41 mmol) in CH2Cl2 (10 mL). After stirring for 18 h, the mixture was
diluted with CH2Cl2, washed with 10% (w/v) aqueous NaCl, and the
organic layer was dried, filtered, and concentrated. The crude residue was
purified by column chromatography (toluene/EtOAc 95:5) to obtain 17
(0.79 g, 96%). Rf ˆ 0.41 (toluene/EtOAc 8:2); [a]2D0 ˆ ‡58 (c ˆ 0.8);
1H NMR (CDCl3): d ˆ 8.09 ± 7.93, 7.50 ± 7.30 (2m, 15H, 3PhCO), 5.53
(dd, J2,3 ˆ 9.8, J3,4 ˆ 8.6 Hz, 1H; H-3), 5.41 (dd, J1,2 ˆ 7.8 Hz, 1H; H-2), 4.76
(d, 1H; H-1), 3.61 (ddd, 1H; OCHHCH2CH2N3), 3.26 ± 3.15 (m, 2H;
OCH2CH2CH2N3), 1.85 ± 1.68 (m, 2H; OCH2CH2CH2N3); 13C NMR
(CDCl3): d ˆ 167.0, 166.9, 165.2 (3PhCO), 101.0 (C-1), 76.1, 74.3, 71.4,
2,3-Di-O-benzoyl-4,6-O-benzylidene-d-glucopyranose (13): Tris(triphenyl-
phosphine)rhodium(i) chloride (2.1 g) was added to a solution of 5 (3.02 g,
5.84 mmol) in EtOH (100 mL) and toluene (250 mL). After stirring at
boiling under reflux for 3 h, TLC (toluene/EtOAc 8:2) showed the
formation of a new spot (Rf ˆ 0.81), and the mixture was concentrated.
The residue was dissolved in THF (250 mL), and water (30 mL) and NIS
(2.0 g) were added. After 20 min, the mixture was concentrated, diluted
with CH2Cl2, washed with 10% (w/v) aqueous NaHSO3 (2 Â ) and 10%
(w/v) aqueous NaCl, and the organic layer was dried, filtered, and
concentrated. The crude residue was purified by column chromatography.
Impurities were eluted with toluene/EtOAc 9:1, and 13 was obtained as a
light brown syrup by elution with toluene/EtOAc 8:2 (2.09 g, 75%; a/b ˆ
3:2). Rf ˆ 0.25 (toluene/EtOAc 8:2); 1H NMR (CDCl3): d ˆ 7.99 ± 7.94,
7.45 ± 7.25 (2m, 15H, PhCH, 2PhCO), 6.14 (t, J2,3 ˆ 9.8, J3,4 ˆ 9.8 Hz, 0.6H;
H-3a), 5.83 (t, J2,3 ˆ 9.6, J3,4 ˆ 9.6 Hz, 0.4H; H-3b), 5.66 (d, J1,2 ˆ 3.7 Hz,
69.4
(C-2,3,4,5),
66.4
(OCH2CH2CH2N3),
63.3
(C-6),
47.7
(OCH2CH2CH2N3), 28.8 (OCH2CH2CH2N3); elemental analysis calcd
(%) for C30H29O9N3 (575.5): C 62.60, H 5.08; found: C 62.68, H 5.14.
3-Azido-1-propanol (18): Sodium azide (3.58 g, 55.0 mmol) was added to a
solution of 3-bromo-1-propanol (1.54 g, 11.1 mmol) in DMF (9 mL). After
stirring at boiling under reflux for 36 h, the mixture was filtered, and co-
concentrated with toluene. The residue was diluted with CH2Cl2 and
washed twice with 10% (w/v) aqueous NaCl. The organic layer was dried,
filtered, and concentrated. An analytically pure sample was obtained by
distillation under reduced pressure (0.83 g, 74%). nD (1.4521, 238C); lit[36]
0.6H; H-1a), 5.54 (s, 0.6H; PhCHa), 5.49 (s, 0.4H; PhCHb), 5.37 (dd, J1,2
ˆ
7.9 Hz, 0.4H; H-2b), 5.30 (dd, 0.6H; H-2a), 4.99 (d, 0.4H; H-1b); 13C NMR
(CDCl3): d ˆ 166.0, 165.7 (2PhCO), 101.4 (PhCHa), 101.3 (PhCHb), 96.1
(C-1a), 91.0 (C-1b), 68.3 (C-6a), 66.6 (C-6b); elemental analysis calcd (%) for
C27H24O8 (476.4): C 68.06, H 5.08; found: C 68.08, H 5.06.
Chem. Eur. J. 2001, 7, No. 20
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0720-4417 $ 17.50+.50/0
4417

D. J. Lefeber et al.
FULL PAPER
(1.4569, 288C); 1H NMR (CDCl3): d ˆ 3.73 (t, 2H; HOCH2CH2CH2N3),
3.44 (t, 2H; HOCH2CH2CH2N3), 1.87 ± 1.79 (m, 2H; HOCH2CH2CH2N3);
IR (KBr; liquid film): nÄ ˆ 2100 cm 1 (N3).
7.91, 7.48 ± 7.26 (2m, 25H; PhCH, 4PhCO), 5.69 (t, J ꢀ9.3 Hz, 1H; H-3),
5.38 (t, J ꢀ9.5 Hz, 1H; H-3'), 5.35, 5.27 (2 dd, J1,2 , J1',2' ˆ 7.6, 7.8 Hz, each
1H; H-2,2'), 5.20 (s, 1H; PhCH), 4.78, 4.65 (2d, each 1H; H-1,1'), 4.07, 3.53
(2t, J ꢀ9.4 Hz, each 1H; H-4,4'), 3.91, 3.86 (2d, each 1H; ClCH2CO), 3.16
(dt, 1H; OCH2CH2CH2N3), 1.77 ± 1.61 (m, 2H; OCH2CH2CH2N3);
13C NMR (CDCl3): d ˆ 166.3, 165.5, 165.1, 164.9, 164.8 (4PhCO,
ClCH2CO), 136.3 (PhCH, quaternary C), 101.5, 101.2, 100.7 (PhCH,
C-1,1'), 67.4 (C-6'), 66.3 (OCH2CH2CH2N3), 62.1 (C-6), 47.7
Allyl (2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-d-glucopyrano-
syl)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside (19): A solution of 7
(0.42 g, 0.80 mmol) and 12 (0.85 g, 1.43 mmol) in dry CH2Cl2 (13 mL),
containing 4 Š molecular sieves, was stirred under Ar for 1 h. Then,
TMSOTf (15 mL, 84 mmol) was added. After 30 min, the mixture was
neutralised with dry pyridine, filtered over cotton, diluted with CH2Cl2, and
washed with 10% (w/v) aqueous NaCl. The organic layer was dried,
filtered, and concentrated. The residue was purified by column chroma-
tography (toluene/EtOAc 95:5) to obtain 19 (0.73 g, 96%). Rf ˆ 0.35
(toluene/EtOAc 9:1); [a]D20 ˆ ‡29 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.01 ±
7.91, 7.50 ± 7.25 (2m, 25H; PhCH, 4PhCO), 5.77 ± 5.64 (m, 1H;
(OCH2CH2CH2N3), 40.2 (ClCH2CO), 28.7 (OCH2CH2CH2N3); IR (KBr,
1
liquid film): nÄ ˆ 2096 cm
(N3); elemental analysis calcd (%) for
C52H48O16ClN3 (1006.4): C 62.05, H 4.80; found: C 62.15, H 4.86.
3-Azidopropyl (2-O-benzoyl-4,6-O-benzylidene-b-d-glucopyranosyl)-
(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside (23): DABCO (0.43 g,
3.8 mmol) was added to a solution of 22 (0.26 g, 0.26 mmol) in toluene
(14 mL) and ethanol (14 mL). After 2.5 h at 558C, the mixture was diluted
with CH2Cl2, washed with aqueous 0.05m HCl, and the organic layer was
dried, filtered, and concentrated. The residue was purified by column
chromatography (toluene/EtOAc 9:1) to afford 23 (0.23 g, 98%). Rf ˆ 0.25
(toluene/EtOAc 9:1); [a]D20 ˆ ‡31 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.08 ±
7.91, 7.50 ± 7.29 (2m, 25H; PhCH, 4 PhCO), 5.68 (t, J2,3 ˆ 9.8 Hz, 1H;
ˆ
OCH2CH CH2), 5.69 (t, J2,3 ˆ 9.7 Hz, 1H; H-3), 5.39 (dd, J1,2 ˆ 7.8 Hz,
1H; H-2), 5.38 (t, J2',3' ˆ J3',4' ˆ 9.4 Hz, 1H; H-3'), 5.27 (dd, J1',2' ˆ 7.6 Hz, 1H;
H-2'), 5.20 (s, 1H; PhCH), 5.16 ± 5.10, 5.07 ± 5.03 (2m, each 1H;
ˆ
OCH2CH CH2), 4.77, 4.71 (2d, each 1H; H-1,1'), 3.91, 3.85 (2d, Jgem
ˆ
14.8 Hz, each 1H; ClCH2CO); 13C NMR (CDCl3): d ˆ 166.3, 165.6, 165.1
(2C), 164.9 (4PhCO, ClCH2CO), 136.2 (PhCH, quaternary C), 117.6
ˆ
ˆ
(OCH2CH CH2), 101.5, 101.1, 99.2 (PhCH, C-1,1'), 69.8 (OCH2CH CH2),
67.3 (C-6'), 62.2 (C-6), 40.2 (ClCH2CO); elemental analysis calcd (%) for
C52H47O16Cl (963.3): C 64.83, H 4.91; found: C 64.59, H 4.97.
H-3), 5.36 (dd, J1,2 ˆ 7.9 Hz, 1H; H-2), 5.22 (s, 1H; PhCH), 5.17 (dd, J1',2'
ˆ
8.0, J2',3' ˆ 9.0 Hz, 1H; H-2'), 4.67, 4.62 (2d, each 1H; H-1,1'), 4.05, 3.36 (2t,
J ꢀ9.3 Hz, each 1H; H-4,4'), 3.87 (t, 1H; H-3'), 3.19 ± 3.11 (m, 2H;
OCH2CH2CH2N3), 1.74 ± 1.60 (m, 2H; OCH2CH2CH2N3); 13C NMR
(CDCl3): d ˆ 165.8, 165.3, 165.1, 165.0 (4PhCO), 136.6 (PhCH, quaternary
C), 101.5, 101.4, 100.7 (PhCH, C-1,1'), 67.5 (C-6'), 66.3 (OCH2CH2CH2N3),
62.4 (C-6), 47.7 (OCH2CH2CH2N3), 28.7 (OCH2CH2CH2N3); elemental
analysis calcd (%) for C50H47O15N3 (929.9): C 64.58, H 5.09; found: C 64.65,
H 5.20.
(2-O-Benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-d-glucopyranosyl)-
(1 ! 4)-2,3,6-tri-O-benzoyl-d-glucopyranose (20): A catalytic amount of
DABCO and tris(triphenylphosphine)rhodium(i) chloride (0.82 g) were
added to a solution of 19 (0.74 g, 0.78 mmol) in absolute EtOH (20 mL),
toluene (45 mL), and CH2Cl2 (3 mL). After stirring at boiling under reflux
for 2.5 h, TLC (toluene/EtOAc 9:1) showed the formation of a new spot
(Rf ˆ 0.38), and the mixture was concentrated. The residue was dissolved in
THF (55 mL), and water (8 mL) and NIS (0.33 g) were added. After
20 min, the mixture was concentrated, diluted with CH2Cl2, washed with
10% (w/v) aqueous NaHSO3 (2 Â ) and 10% (w/v) aqueous NaCl, and the
organic layer was dried, filtered, and concentrated. The crude residue was
purified by column chromatography. Impurities were eluted with toluene/
EtOAc 9:1, and 20 was obtained as a light brown syrup by elution with
toluene/EtOAc 8:2 (0.50 g, 70%; a/b ˆ 4:1). Rf ˆ 0.29a/0.34b (toluene/
EtOAc 8:2); 1H NMR (CDCl3): d ˆ 8.06 ± 7.93, 7.50 ± 7.31 (2m, 25H; PhCH,
3-Azidopropyl (2-O-benzoyl-b-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-ben-
zoyl-b-d-glucopyranoside (24): CF3COOH (60 mL) and H2O (8 mL) were
added to a solution of 23 (80 mg, 87 mmol) in CH2Cl2 (5 mL). The mixture
was stirred for 3 h, then diluted with CH2Cl2, washed with 10% (w/v)
aqueous NaHCO3 (2 Â ) and 10% (w/v) aqueous NaCl, and the organic
layer was dried, filtered, and concentrated. The residue was purified by
column chromatography (toluene/EtOAc 8:2 ! toluene/EtOAc 6:4) to
obtain 24 (70 mg, 90%). Rf ˆ 0.04 (toluene/EtOAc 6:4); [a]2D0 ˆ ‡28 (c ˆ
1); 1H NMR (CDCl3): d ˆ 8.04 ± 7.90, 7.61 ± 7.32 (2m, 20H; 4 PhCO), 5.66 (t,
J2,3 ˆ 9.7 Hz, 1H; H-3), 5.38 (dd, J1,2 ˆ 7.8 Hz, 1H; H-2), 4.97 (dd, J1',2' ˆ 7.8,
J2',3' ˆ 9.5 Hz, 1H; H-2'), 4.67, 4.65 (2d, each 1H; H-1,1'), 3.52 (ddd, 1H;
OCHHCH2CH2N3), 3.19 ± 3.14 (m, 2H; OCH2CH2CH2N3), 1.79 ± 1.64 (m,
2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ 165.9 (3C), 165.2
(4PhCO), 100.7 (C-1,1'), 66.4 (OCH2CH2CH2N3), 62.6, 61.3 (C-6,6'), 47.7
(OCH2CH2CH2N3), 28.8 (OCH2CH2CH2N3); elemental analysis calcd (%)
for C43H43O15N3 (841.3): C 61.35, H 5.14; found: C 61.59, H 5.22.
4PhCO), 6.04 (dd, J2,3 ˆ 10.2, J3,4 ˆ 9.2 Hz, 0.8H; H-3a), 5.48 (d, J1,2
ˆ
3.5 Hz, 0.8H; H-1a), 5.42 (t, J2',3' ˆ J3',4' ˆ 9.4 Hz, 1H; H-3'), 5.30 (dd,
J1',2' ˆ 7.6 Hz, 1H; H-2'), 5.24 (s, 0.8H; PhCHa), 5.22 (s, 0.2H; PhCHb), 5.14
(dd, 0.8H; H-2a), 4.89, 4.81 (2d, J1,2 ꢀ7.8 Hz, each 0.2H; H-1b,1'b), 4.85 (d,
0.8H; H-1'a), 3.93, 3.87 (2d, Jgem ˆ 14.9 Hz, each 1H; ClCH2CO); 13C NMR
(CDCl3): d ˆ 166.7, 166.4, 165.8, 165.0 (2C) (4PhCO, ClCH2CO), 136.3
(PhCH, quaternary C), 101.6, 101.2 (PhCH, C-1'), 95.4 (C-1b), 90.0 (C-1a),
67.4 (C-6'), 62.0 (C-6), 40.3 (ClCH2CO); elemental analysis calcd (%) for
C49H43O16Cl (923.3): C 63.74, H 4.69; found: C 63.44, H 4.73.
3-Azidopropyl (2-O-benzoyl-b-d-glucopyranosyluronic acid)-(1 ! 4)-2,3,6-
tri-O-benzoyl-b-d-glucopyranoside (25): A catalytic amount of TEMPO,
and a solution (0.14 mL) of KBr (7.8 mg) and Bu4NBr (10.4 mg) in
saturated aqueous NaHCO3 (1.4 mL) were added to a solution of 24
(65 mg, 0.072 mmol) in CH2Cl2 (0.65 mL). The mixture was stirred
vigorously at 08C, when a solution of saturated aqueous NaCl (0.14 mL),
saturated aqueous NaHCO3 (78 mL), and aqueous NaOCl (13% Cl active;
0.18 mL) was added dropwise. After 45 min, the mixture was acidified with
4m HCl (pH 4), diluted with CH2Cl2, and the organic layer was washed with
10% (w/v) aqueous NaCl, dried, filtered, and concentrated. The residue
was purified by column chromatography. Impurities were eluted with
CH2Cl2/acetone 8:2, and 25 (55 mg, 85%) was eluted with CH2Cl2/acetone/
HOAc 8:2:0.5. Rf ˆ 0.30 (CH2Cl2/acetone/HOAc 8:2:1); 13C NMR
(CDCl3): d ˆ 100.6, 100.4 (C-1,1'), 66.3 (OCH2CH2CH2N3), 62.3 (C-6),
47.6 (OCH2CH2CH2N3), 28.7 (OCH2CH2CH2N3).
(2-O-Benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-d-glucopyranosyl)-
(1 ! 4)-2,3,6-tri-O-benzoyl-a-d-glucopyranosyl trichloroacetimidate (21):
Cl3CCN (0.60 mL) and DBU (18 mL) were added to a solution of 20 (0.40 g,
0.44 mmol) in dry CH2Cl2 (9 mL). After 2 h, the mixture was concentrated
and the residue was purified by column chromatography (toluene/EtOAc
95:5) to yield 21 (0.41 g, 88%). Rf ˆ 0.32 (toluene/EtOAc 9:1); 1H NMR
(CDCl3): d ˆ 8.54 (s, 1H; NH), 8.05 ± 7.90, 7.49 ± 7.25 (2m, 25H; 4PhCO,
PhCH), 6.67 (d, J1,2 ˆ 3.7 Hz, 1H; H-1), 6.08 (dd, J2,3 ˆ 10.1, J3,4 ˆ 8.8 Hz,
1H; H-3), 5.46 (dd, 1H; H-2), 5.41 (t, J2',3' ˆ 9.2 Hz, 1H; H-3'), 5.23 (dd,
J1',2' ˆ 7.6 Hz, 1H; H-2'), 5.28 (s, 1H; PhCH), 4.87 (d, 1H; H-1'), 3.91, 3.85
(2d, Jgem ˆ 14.8 Hz, each 1H; ClCH2CO); 13C NMR (CDCl3): d ˆ 166.3,
165.4, 165.3, 164.9, 164.8 (4PhCO, ClCH2CO), 160.4 (OC(NH)CCl3), 136.3
(PhCH, quaternary C), 101.7, 101.2 (PhCH, C-1'), 92.8 (C-1), 67.5 (C-6'),
61.6 (C-6), 40.2 (ClCH2CO).
A small amount was methylated with diazomethane and acetylated with
Ac2O and pyridine for analysis. 1H NMR (CDCl3): d ˆ 7.94 ± 7.89, 7.50 ± 7.26
(2m, 20H; 4PhCO), 4.87, 4.65 (2d, J1,2 , J1',2' ˆ 7.4, 7.7 Hz, each 1H; H-1,1'),
3.73 (d, J4',5' ˆ 9.9 Hz, 1H; H-5'), 3.37 (s, 3H; COOCH3), 3.16 ± 3.13 (m, 2H;
OCH2CH2CH2N3), 1.93, 1.84 (2s, each 3H; CH3CO), 1.75 ± 1.57 (m, 2H;
OCH2CH2CH2N3); elemental analysis calcd (%) for C48H47O18N3 (953.9): C
60.44, H 4.97; found: C 60.65, H 5.03.
3-Azidopropyl (2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-d-glu-
copyranosyl)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside (22): A sol-
ution of 12 (0.50 g, 0.84 mmol) and 17 (0.28 g, 0.49 mmol) in dry CH2Cl2
(10 mL), containing 4 Š molecular sieves, was stirred under Ar for 1 h.
Then, TMSOTf (11.5 mL, 63 mmol) was added. After 30 min, the mixture
was neutralised with dry pyridine, filtered over cotton, diluted with CH2Cl2,
and washed with 10% (w/v) aqueous NaCl. The organic layer was dried,
filtered, and concentrated. The residue was purified by column chroma-
tography (toluene/EtOAc 95:5) to obtain 22 (0.38 g, 78%). Rf ˆ 0.39
(toluene/EtOAc 9:1); [a]D20 ˆ ‡42 (c ˆ 1); 1H NMR (CDCl3): d ˆ 8.06 ±
3-Aminopropyl (b-d-glucopyranosyluronic acid)-(1 ! 4)-b-d-glucopyrano-
side (1): NaOMe was added until pH 11 to a solution of 25 (90 mg,
0.11 mmol) in MeOH (9 mL). After stirring for 1.5 h, TLC (EtOAc/MeOH/
4418
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0720-4418 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 20

Oligosaccharides
4411±4421
H2O 12:5:3) showed the formation of a new spot (Rf ˆ 0.37), and the
3.62 (d, J4',5' ˆ 9.3 Hz, 1H; H-5'), 3.12 (dt, 2H; OCH2CH2CH2N3), 1.71 ± 1.59
(m, 2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ 169.1 (C-6'), 166.0,
165.7, 165.5, 165.3, 165.1, 164.9, 164.7, 163.8 (8PhCO), 101.4, 100.7, 100.6
(C-1,1',1''), 66.3 (OCH2CH2CH2N3), 62.5, 62.1 (C-6,6''), 47.7
(OCH2CH2CH2N3), 28.8 (OCH2CH2CH2N3); MS (MALDI-TOF): m/z:
‡
mixture was neutralised with Dowex H , filtered, and concentrated. A
solution of the residue in water was washed with CH2Cl2 (3 Â ), and the
aqueous layer was concentrated. MALDI-TOF analysis of the residue
‡
showed a peak at m/z 462 [M‡Na] . Then, a solution of the residue in 0.1m
‡
NaOH (1.1 mL) was added dropwise to a suspension of 10% Pd/C (2 mg)
and NaBH4 (8.7 mg) in bidistilled water (0.55 mL). After 45 min, when
TLC (EtOAc/MeOH/H2O 12:5:3) showed the formation of a ninhydrin-
1456 [M‡Na] .
3-Aminopropyl (b-d-glucopyranosyl)-(1 ! 3)-(b-d-glucopyranosyluronic
acid)-(1 ! 4)-b-d-glucopyranoside (2): NaOMe was added until pH 11 to
a solution of 29 (50 mg, 33 mmol) in MeOH (3 mL). After stirring for 16 h,
TLC (EtOAc/MeOH/H2O 12:5:2) showed the formation of a new spot
‡
positive spot on the baseline, the mixture was acidified with Dowex H
‡
(pH 4), then loaded on a short column of Dowex 50 WÂ 2 (H , 200 ±
400 mesh). After elution of contaminants with water, elution with 1%
‡
(Rf ˆ 0.27), and the mixture was neutralised with Dowex H , filtered, and
NH4OH afforded 1 after concentration and co-concentration with water
concentrated. A solution of the residue in water was washed with CH2Cl2
(2 Â ) (39 mg, 90%). [a]D20
ˆ
92 (c ˆ 0.6, H2O); MS (MALDI-TOF): m/z:
(3 Â ), and the aqueous layer was concentrated. MALDI-TOF analysis of
‡
‡
1
414 [M‡H] , 436 [M‡Na] . For H NMR data, see Table 1.
‡
the residue showed a peak at m/z 624 [M‡Na] . Then, a solution of the
residue in 0.1m NaOH (0.33 mL) was added dropwise to a suspension of
10% Pd/C (0.6 mg) and NaBH4 (2.7 mg) in bidistilled water (0.17 mL).
After 45 min, when TLC (EtOAc/MeOH/H2O 12:5:3) showed the for-
mation of a ninhydrin-positive spot on the baseline, the mixture was
3-Azidopropyl (2,3,4,6-tetra-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-
benzoyl-4,6-O-benzylidene-b-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-ben-
zoyl-b-d-glucopyranoside (27): A solution of 23 (53 mg, 57 mmol) and ethyl
2,3,4,6-tetra-O-benzoyl-1-thio-b-d-glucopyranoside (26; 62 mg, 96 mmol) in
dry CH2Cl2 (0.26 mL), containing 4 Š molecular sieves, was stirred under
Ar for 30 min at 08C. Then, 0.48 mL of a solution of NIS (420 mg,
1.87 mmol) and TfOH (12 mL) in Et2O/CH2Cl2 (1:1) (9.6 mL) was added.
After 5 min, the mixture was neutralised with dry pyridine, filtered over
cotton, diluted with CH2Cl2, and washed with 10% (w/v) aqueous NaHSO3
(2 Â ) and 10% (w/v) aqueous NaCl. The organic layer was dried, filtered,
and concentrated. The residue was purified by column chromatography
(toluene/EtOAc 9:1) to afford 27 (70 mg, 82%). Rf ˆ 0.41 (toluene/EtOAc
85:15); [a]2D0 ˆ ‡27 (c ˆ 1); 1H NMR (CDCl3): d ˆ 5.64, 5.56 (2t, J2'',3'' ˆ 9.3,
J3'',4'' ˆ 9.4 Hz, each 1H; H-3'',4''), 5.62 (dd, J2,3 ˆ 9.8, J3,4 ˆ 9.0 Hz, 1H; H-3),
5.39, 5.31 (2dd, J1,2 , J1'',2'' ˆ 7.8 Hz, each 1H; H-2,2''), 5.29 (s, 1H; PhCH),
5.27 (dd, J1',2' ˆ 7.8, J2',3' ˆ 9.0 Hz, 1H; H-2'), 4.91 (d, 1H; H-1''), 4.59, 4.58
(2d, each 1H; H-1,1'), 3.47 (ddd, 1H; OCHHCH2CH2N3), 3.13 (dt, 2H;
OCH2CH2CH2N3), 1.72 ± 1.58 (m, 2H; OCH2CH2CH2N3); 13C NMR
(CDCl3): d ˆ 165.9, 165.1, 164.9, 164.8 (2C), 164.6, 164.0 (2C) (8PhCO),
136.6 (PhCH, quaternary C), 101.5, 101.2, 100.5 (2C) (PhCH, C-1,1',1''),
67.5 (C-6'), 66.3 (OCH2CH2CH2N3), 62.8 (2C) (C-6,6''), 47.7
(OCH2CH2CH2N3), 28.7 (OCH2CH2CH2N3); MS (MALDI-TOF): m/z:
‡
acidified with Dowex H (pH 4), then loaded on a short column of Dowex
‡
50 WÂ 2 (H , 200 ± 400 mesh). After elution of contaminants with water,
elution with 1% NH4OH afforded 2 (17 mg, 89%), after concentration and
co-concentration with water (2 Â ). [a]D20
ˆ
18 (c ˆ 0.8, H2O); MS
‡
‡
(MALDI-TOF): m/z: 575 [M‡H] , 597 [M‡Na] . For 1H NMR data,
see Table 1.
3-Azidopropyl (2-O-benzoyl-4,6-O-benzylidene-3-O-chloroacetyl-b-d-glu-
copyranosyl)-(1 ! 4)-(2,3,6-tri-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-
O-benzoyl-4,6-O-benzylidene-b-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-
benzoyl-b-d-glucopyranoside (30): A solution of 21 (0.50 g, 0.47 mmol) and
23 (0.27 g, 0.29 mmol) in dry CH2Cl2 (4.4 mL), containing 4 Š molecular
sieves, was stirred under Ar for 1 h. Then, TMSOTf (8.5 mL, 47 mmol) was
added. After 15 min, the mixture was neutralised with dry pyridine, filtered
over cotton, diluted with CH2Cl2, and washed with 10% (w/v) aqueous
NaCl. The organic layer was dried, filtered, and concentrated. The residue
was purified by column chromatography (toluene/EtOAc 95:5) to obtain 30
(0.39 g, 75%). Rf ˆ 0.36 (toluene/EtOAc 85:15); [a]2D0 ˆ ‡34 (c ˆ 1);
1H NMR (CDCl3): d ˆ 5.58, 5.40, 5.25 (3t,
J
ꢀ9.5 Hz, each 1H;
‡
1530 [M‡Na] .
H-3,3'',3'''), 5.17, 5.11 (2s, each 1H; 2 PhCH), 4.72, 4.55, 4.53, 4.53 (4d, J
ꢀ7.7 Hz, each 1H; H-1,1',1'',1'''), 3.87, 3.81 (2d, Jgem ˆ 14.9 Hz, each 1H;
ClCH2CO), 1.79 ± 1.65 (m, 2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ
136.4, 136.2 (2PhCH, quaternary C), 101.4 (2C), 101.1 (2C), 100.6, 99.8
(2PhCH, C-1,1',1'',1'''), 67.5, 67.3 (C-6',6'''), 66.3 (OCH2CH2CH2N3), 62.0,
61.9 (C-6,6''), 47.7 (OCH2CH2CH2N3), 40.2 (ClCH2CO), 28.7
3-Azidopropyl (2,3,4,6-tetra-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-
benzoyl-b-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyra-
noside (28): CF3COOH (60 mL) and H2O (8 mL) were added to a solution of
27 (0.13 g, 82 mmol) in CH2Cl2 (5 mL). The mixture was stirred for 3 h,
diluted with CH2Cl2, washed with 10% (w/v) aqueous NaHCO3 (2 Â ), and
the organic layer was dried, filtered, and concentrated. The residue was
purified by column chromatography (toluene/EtOAc 8:2 ! toluene/
EtOAc 7:3) to obtain 28 (86 mg, 70%). Rf ˆ 0.37 (toluene/EtOAc 7:3);
‡
‡
(OCH2CH2CH2N3); MS (FAB): m/z: 1834 [M‡H] , 1856 [M‡Na] .
3-Azidopropyl (2-O-benzoyl-4,6-O-benzylidene-b-d-glucopyranosyl)-
(1 ! 4)-(2,3,6-tri-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-benzoyl-
4,6-O-benzylidene-b-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-
glucopyranoside (31): Thiourea (50 mg) was added to a solution of 30
(0.39 g, 0.22 mmol) in ethanol (5 mL) and pyridine (0.66 mL). After stirring
for 3 h at 908C, the mixture was cooled to room temperature, co-
concentrated with toluene, and the residue was purified by column
chromatography (toluene/EtOAc 95:5 ! toluene/EtOAc 9:1) to afford 31
(0.32 g, 84%). Rf ˆ 0.25 (toluene/EtOAc 8:2); [a]2D0 ˆ ‡52 (c ˆ 1); 1H NMR
(CDCl3): d ˆ 5.58, 5.39 (2t, J ꢀ9.5 Hz, each 1H; H-3,3''), 5.17, 5.13 (2s, each
1H; 2PhCH), 4.73, 4.54 (2H), 4.49 (3d, J ꢀ7.8 Hz, 4H; H-1,1',1'',1'''), 3.11
(brt, 2H; OCH2CH2CH2N3), 1.70 ± 1.59 (m, 2H; OCH2CH2CH2N3);
13C NMR (CDCl3): d ˆ 165.9, 165.6, 165.2, 165.1, 164.8 (2C), 164.7, 163.9
(8PhCO), 136.4 (2PhCH, quaternary C), 101.4 (3C), 101.1, 100.6, 100.0
(2PhCH, C-1,1',1'',1'''), 67.4 (2C) (C-6',6'''), 66.3 (OCH2CH2CH2N3), 62.1
(2C) (C-6,6''), 47.7 (OCH2CH2CH2N3), 28.7 (OCH2CH2CH2N3); MS
1
[a]2D0 ˆ ‡14 (c ˆ 1); H NMR (CDCl3): d ˆ 5.76, 5.54, 5.53 (3t, J ꢀ9.5 Hz,
each 1H; H-3,3'',4''), 5.46 (dd, J1'',2'' ˆ 7.9, J2'',3'' ˆ 9.8 Hz, 1H; H-2''), 5.35 (dd,
J1,2 ˆ 7.7, J2,3 ˆ 9.6 Hz, 1H; H-2), 5.14 (dd, J1',2' ˆ 8.0, J2',3' ˆ 9.2 Hz, 1H;
H-2'), 4.81 (d, 1H; H-1''), 4.56 (d, 1H; H-1), 4.51 (d, 1H; H-1'), 3.14 (dt,
2H; OCH2CH2CH2N3), 1.75 ± 1.62 (m, 2H; OCH2CH2CH2N3); 13C NMR
(CDCl3): d ˆ 165.9, 165.5, 165.1 (2C), 164.9, 164.7, 163.8, 160.2 (8PhCO),
101.5, 100.8 (2C) (C-1,1',1''), 66.3 (OCH2CH2CH2N3), 62.5, 62.2 (2C)
(C-6,6',6''), 47.7 (OCH2CH2CH2N3), 28.8 (OCH2CH2CH2N3); MS (MAL-
‡
DI-TOF): m/z: 1442 [M‡Na] .
3-Azidopropyl (2,3,4,6-tetra-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-
benzoyl-b-d-glucopyranosyluronic acid)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-
glucopyranoside (29): TEMPO (catalytic amount), and a solution (70 mL)
of KBr (3.6 mg) and Bu4NBr (4.9 mg) in saturated aqueous NaHCO3
(0.7 mL) were added to a solution of 28 (50 mg, 34 mmol) in CH2Cl2
(0.6 mL). The mixture was stirred vigorously at 08C, when a solution of
saturated aqueous NaCl (70 mL), saturated aqueous NaHCO3 (33 mL), and
aqueous NaOCl (13% Cl active; 90 mL) was added dropwise. After 45 min,
the mixture was acidified with 4m HCl (pH 4), diluted with CH2Cl2, and the
organic layer was washed with 10% (w/v) aqueous NaCl, dried, filtered,
and concentrated. The residue was purified by column chromatography.
Impurities were eluted with CH2Cl2/acetone (8:2), and 29 (38 mg, 76%)
was eluted with CH2Cl2/acetone/HOAc (8:2:0.5). Rf ˆ 0.65 (CH2Cl2/ace-
tone/HOAc 8:2:1); [a]2D0 ˆ ‡25 (c ˆ 1); 1H NMR (CDCl3): d ˆ 5.76, 5.64,
5.53 (3t, J2,3 ˆ 9.6, J2'',3'' ˆ 9.8, J3'',4'' ˆ 9.3 Hz, each 1H; H-3,3'',4''), 5.45 (dd,
‡
‡
(FAB): m/z: 1758 [M‡H] , 1780 [M‡Na] .
3-Azidopropyl
(2-O-benzoyl-b-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-
benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-benzoyl-b-d-glucopyranosyl)-
(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside
(32):
CF3COOH
(0.12 mL) and H2O (10 mL) were added to a solution of 31 (0.20 g,
0.11 mmol) in CH2Cl2 (6.5 mL). The mixture was stirred for 2.5 h, diluted
with CH2Cl2, washed with 10% (w/v) aqueous NaHCO3 (2 Â ) and 10%
(w/v) aqueous NaCl, and the organic layer was dried, filtered, and
concentrated. The residue was purified by column chromatography
(toluene/EtOAc 1:1 ! toluene/EtOAc 3:7) to obtain 32 (0.12 g, 70%).
Rf ˆ 0.14 (toluene/EtOAc 1:1); [a]2D0 ˆ ‡36 (c ˆ 1); 1H NMR (CDCl3): d ˆ
5.52 (brt, J ꢀ9.3 Hz, 2H; H-3,3''), 5.31, 5.29, 5.05, 4.91 (4dd, each 1H;
J1'',2'' ˆ 7.9 Hz, 1H; H-2''), 5.27 (dd, J1,2 ˆ 7.7 Hz, 1H; H-2), 5.18 (dd, J1',2'
ˆ
7.9 Hz, 1H; H-2'), 4.86 (d, 1H; H-1''), 4.68 (d, 1H; H-1'), 4.58 (d, 1H; H-1),
Chem. Eur. J. 2001, 7, No. 20
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0720-4419 $ 17.50+.50/0
4419

D. J. Lefeber et al.
FULL PAPER
H-2,2',2'',2'''), 4.63, 4.61, 4.54, 4.45 (4d, J ꢀ7.5 Hz, each 1H; H-1,1',1'',1'''),
1.68 ± 1.56 (m, 2H; OCH2CH2CH2N3); 13C NMR (CDCl3): d ˆ 165.9, 165.8,
165.7, 165.1 (2C), 165.0, 164.9, 163.9 (8PhCO), 101.2, 100.7 (2C), 100.6 (C-
1,1',1'',1'''), 66.3 (OCH2CH2CH2N3), 62.3 (2C), 62.0, 61.3 (C-6,6',6'',6'''), 47.7
(OCH2CH2CH2N3), 28.8 (OCH2CH2CH2N3); MS (FAB): m/z: 1582
concentration, the residue was purified by column chromatography
(toluene/EtOAc 7:3). To a solution of the residue in MeOH (1 mL) and
H2O (0.5 mL) was added 0.5m NaOH until pH 10 was reached. After
‡
stirring overnight, Dowex H was added until pH 6, and the mixture was
filtered and concentrated to obtain 36. A solution of the residue in 0.1m
NaOH (1 mL) was added dropwise to a suspension of 10% Pd/C (2 mg)
and NaBH4 (8.1 mg) in H2O (0.5 mL). After 30 min, when TLC (EtOAc/
MeOH/H2O 12:5:2) showed the formation of 37 as a ninhydrin-positive
‡
‡
[M‡H] , 1604 [M‡Na] .
3-Azidopropyl (2-O-benzoyl-b-d-glucopyranosyluronic acid)-(1 ! 4)-
(2,3,6-tri-O-benzoyl-b-d-glucopyranosyl)-(1 ! 3)-(2-O-benzoyl-b-d-gluco-
‡
spot on the baseline, the mixture was acidified with Dowex H (pH 4) and
pyranosyluronic
acid)-(1 ! 4)-2,3,6-tri-O-benzoyl-b-d-glucopyranoside
‡
loaded on a short column of Dowex 50 WÂ 2 (H , 200 ± 400 mesh).
(33): TEMPO (catalytic amount), and a solution (0.12 mL) of KBr
(6.6 mg) and Bu4NBr (8.7 mg) in saturated aqueous NaHCO3 (1.2 mL)
were added to a solution of 32 (95 mg, 57 mmol) in CH2Cl2 (0.6 mL). The
mixture was stirred vigorously at 08C, then a mixture of saturated aqueous
NaCl (0.12 mL), saturated aqueous NaHCO3 (0.06 mL), and aqueous
NaOCl (13% Cl active; 0.15 mL) was added dropwise. After 45 min, the
mixture was acidified with 4m HCl (pH 4), diluted with CH2Cl2, and the
organic layer was washed with 10% (w/v) aqueous NaCl, dried, filtered,
and concentrated. The residue was purified by column chromatography.
Impurities were eluted with CH2Cl2/acetone (8:2), and 33 (64 mg, 65%)
was eluted with CH2Cl2/acetone/HOAc (8:2:0.5). Rf ˆ 0.41 (CH2Cl2/ace-
tone/HOAc 8:2:1).
Contaminants were eluted with water, and after elution with 1% NH4OH,
37 was obtained after concentration and co-concentration with water (2 Â )
(22 mg, 80%). [a]D20
[M‡H] , 274 [M‡Na] . For H NMR data, see Table 1.
ˆ
25 (c ˆ 1, H2O); MS (MALDI-TOF): m/z: 252
‡
‡
1
General procedure for the elongation of 1, 2, 3, 35 and 37 with diethyl
squarate: A solution of diethyl squarate (1 mmol) in EtOH (76.2 mL, stock
solution of 24.9 mL diethyl squarate in 12.8 mL EtOH) was added to a
solution of 3-aminopropyl glycoside (1 mmol) in 0.1m sodium phosphate
(40 mL for 35 and 37, 75 mL for 1 ± 3; pH 6.9). After stirring for 16 h, EtOH
was evaporated by flushing with N2. To the water layer was added H2O (35,
4 mL) or aqueous HOAc pH 4 (1 ± 3, 37, 6 mL), and the mixture was loaded
on a C18 Bakerbond spe column (500 mg). Rinsing with H2O (10 mL)
eluted the starting material, and elution with MeOH (4 mL) and
evaporation of the solvent by flushing with N2, afforded the pure elongated
fragment that was used for the conjugation reaction.
1
A small sample was methylated with diazomethane for analysis. H NMR
(CDCl3): d ˆ 5.56 (brt, J ꢀ9.3 Hz, 2H; H-3,3''), 5.36, 5.28, 5.22, 5.13 (4dd,
each 1H; H-2,2',2'',2'''), 4.84, 4.66, 4.58, 4.54 (4d, J ꢀ7.8 Hz, each 1H;
H-1,1',1'',1'''), 3.39, 3.34 (2s, each 3H; 2COOCH3), 3.11 (dt, 2H;
OCH2CH2CH2N3), 1.68 ± 1.56 (m, 2H; OCH2CH2CH2N3); MS (MALDI-
General procedure for the conjugation of the elongated saccharides to a
protein carrier
‡
‡
TOF): m/z: 1610 [M‡H] , 1632 [M‡Na] .
CRM197-conjugates: For a targeted oligosaccharide incorporation of about
11 mol mol 1, the elongated saccharide fragment (ꢀ1 mmol) was dissolved
in 0.1m sodium borate buffer (400 mL; pH 9.5), and a solution of CRM197
(61.15 mgmL 1; 86 mL, 0.09 mmol) was added. After stirring for two to three
days, the mixture was diluted with H2O to a protein concentration of
1 mgmL 1, samples were taken for MALDI-TOF analysis, and the mixture
was dialysed against 50mm sodium phosphate buffer (pH 7.2). To obtain
different oligosaccharide loadings, the amount of elongated saccharide was
varied. For MALDI-TOF analysis, samples were mixed on the target plate
in a ratio of 1:1 (v/v) with the matrix 3,5-dimethoxy-4-hydroxycinnamic
acid in 30% aqueous CH3CN containing 0.3% CF3COOH.
3-Aminopropyl (b-d-glucopyranosyluronic acid)-(1 ! 4)-(b-d-glucopyra-
nosyl)-(1 ! 3)-(b-d-glucopyranosyluronic acid)-(1 ! 4)-b-d-glucopyrano-
side (3): NaOMe was added until pH 11 to a solution of 33 (75 mg,
43 mmol) in MeOH (3 mL). After stirring for 16 h, TLC (EtOAc/MeOH/
H2O 12:5:3) showed the formation of a new spot (Rf ˆ 0.30), and the
‡
mixture was neutralised with Dowex H , filtered, and concentrated. A
solution of the residue in water was washed with CH2Cl2 (3 Â ), and the
aqueous layer was concentrated. MALDI-TOF analysis of the residue
‡
showed a peak at m/z 800 [M‡Na] . Then, a solution of the residue in 0.1m
NaOH (0.43 mL) was added dropwise to a suspension of 10% Pd/C
(0.8 mg) and NaBH4 (3.5 mg) in bidistilled water (0.22 mL). After 45 min,
KLH-conjugates: The elongated saccharide fragment (ꢀ1 mmol) was
dissolved in 0.1m sodium borate buffer (400 mL; pH 9.5), and a solution
of KLH (50 mgmL 1; 75 mL for 1, 60 mL for 2, and 40 mL for 3) was added.
After stirring for two to three days, the mixture was diluted with 50mm
sodium phosphate buffer (pH 7.2) and dialysed against the same buffer.
when TLC (EtOAc/MeOH/H2O 12:5:3) showed the formation of
a
ninhydrin-positive spot on the baseline, the mixture was acidified with
‡
‡
Dowex H (pH 4), then loaded on a short column of Dowex 50 WÂ 2 (H ,
200 ± 400 mesh). After elution of contaminants with water, elution with 1%
NH4OH afforded 3 after concentration and co-concentration with water
(2  ) (19 mg, 60%). [a]2D0 ˆ ‡124 (c ˆ 0.7, H2O); MS (MALDI-TOF): m/z:
TT-conjugates: The elongated saccharide fragment (ꢀ1 mmol) was dis-
solved in 0.1m sodium borate buffer (300 mL; pH 9.5), and a solution of TT
(6.12 mgmL 1; 0.61 mL for 1, 0.49 mL for 2, and 0.33 mL for 3) was added.
The pH was adjusted to 9.5 by addition of 0.1m NaOH. After stirring for
two to three days, the reaction mixture was diluted with 50mm sodium
phosphate buffer (pH 7.2) and dialysed against the same buffer.
‡
‡
1
752 [M‡H] , 774 [M‡Na] . For H NMR data, see Table 1.
3-Aminopropyl b-d-glucopyranoside (35): NaOMe was added until pH 10
to a solution of 16 (81 mg, 0.12 mmol) in MeOH (3 mL). After stirring for
2 h, when TLC (EtOAc/MeOH/H2O 6:3:1) showed the formation of 34
‡
(Rf ˆ 0.63), the mixture was neutralised with Dowex H , filtered, and
The protein concentrations of all conjugates were determined by the Pierce
assay[34] and the carbohydrate content by MALDI-TOF (CRM197) or
Dubois (KLH and TT) analysis.[33] Coupling efficiencies (Tables 2 and 3)
were expressed as percentage of the targeted incorporation.
concentrated. A solution of the residue in water was washed with CH2Cl2
(3 Â ), then the aqueous layer was concentrated, and a solution of the
residue (crude 34) in 0.1m NaOH (1.1 mL) was added dropwise to a
suspension of 10% Pd/C (2 mg) and NaBH4 (9.7 mg) in bidistilled water
(0.5 mL). After 45 min, when TLC (EtOAc/MeOH/H2O 12:5:3) showed
the formation of 35 as a ninhydrin-positive spot on the baseline, the mixture
‡
was acidified with Dowex H (pH 4) and loaded on a short column of
‡
Acknowledgement
Dowex 50 WÂ 2 (H , 200 ± 400 mesh). Contaminants were eluted with
water, and after elution with 1% NH4OH, 35 was obtained after
concentration and co-concentration with water (2 Â ) (25 mg, 88%).
This work was supported by the European Union program VACNET
(grant ERB BIO 4CT960158). We thank Dr. P. Hoogerhout for the kind gift
of tetanus toxoid.
‡
[a]2D0 ˆ ‡6 (c ˆ 0.2, H2O); MS (MALDI-TOF): m/z: 238 [M‡H] , 260
‡
1
[M‡Na] . For H NMR data, see Table 1.
3-Aminopropyl b-d-glucopyranosiduronic acid (37): TEMPO (0.12 mg)
and KBr (5.6 mg) were added to a cooled solution (58C) of 34 (30 mg,
0.11 mmol) in H2O (3 mL). Aqueous NaOCl (13% Cl active; 0.37 mL) was
brought to pH 10 by addition of 4m HCl and cooled to 58C. The two
solutions were combined and pH 10 was maintained by addition of 0.5m
NaOH. After stirring for 3 h at 58C, the mixture was neutralised with 4m
HCl and concentrated. Desalting of the mixture could not be achieved by
size-exclusion chromatography. Therefore, the crude residue was acety-
lated by stirring with Ac2O (1 mL) and pyridine (1 mL) for 4 h, and after
[1] J. C. Butler, S. F. Dowell, R. F. Breiman, Vaccine 1998, 16, 1693 ± 1697.
[2] J. B. Robbins, R. Austrian, C.-J. Lee, S. C. Rastogi, G. Schiffman, J.
Henrichsen, P. H. Mäkela, C. V. Broome, R. R. Facklam, R. H.
Tiesjema, J. C. Parke, Jr., J. Infect. Dis. 1983, 148, 1136 ± 1159.
[3] C.-J. Lee, T. R. Wang, Crit. Rev. Microbiol. 1994, 20, 1 ± 12.
[4] E. Alonso de Velasco, A. F. M. Verheul, J. Verhoef, H. Snippe,
Microbiol. Rev. 1995, 59, 591 ± 603.
4420
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0947-6539/01/0720-4420 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 20

Oligosaccharides
4411±4421
[5] R. W. Ellis, D. M. Granoff, Development and Clinical Uses of
Haemophilus b Conjugate Vaccines, New York, Dekker, 1994.
[6] P. Richmond, R. Borrow, E. Miller, S. Clark, F. Sadler, A. Fox, N.
Begg, R. Morris, K. Cartwright, J. Infect. Dis. 1999, 179, 1569 ± 1572.
[7] M. B. Rennels, K. M. Edwards, H. L. Keyserling, K. S. Reisinger,
D. A. Hogerman, D. V. Madore, I. Chang, P. R. Paradiso, F. J.
Malinoski, A. Kimura, Pediatrics 1998, 101, 604 ± 611.
[8] H. Snippe, W. T. M. Jansen, J. P. Kamerling, D. J. Lefeber, Clin. Diagn.
Lab. Immunol. 2000, 7, 325.
[22] E. J. Corey, J. Suggs, J. Org. Chem. 1973, 38, 3224.
[23] T. Nukada, H. Lucas, P. Konradson, C. A. A. van Boeckel, Synlett
1991, 11, 365 ± 367.
[24] R. R. Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem. 1984, 1343 ±
1357.
[25] A. E. J. de Nooy, A. C. Bezemer, H. van Bekkum, Synthesis 1996,
1153 ± 1174.
[26] A. M. P. van Steijn, J. P. Kamerling, J. F. G. Vliegenthart, J. Carbohydr.
Chem. 1992, 11, 665 ± 689.
[9] W. T. M. Jansen, S. Hogenboom, M. J. L. Thijssen, J. P. Kamerling,
J. F. G. Vliegenthart, J. Verhoef, H. Snippe, A. F. M. Verheul, Infect.
Immun. 2001, 69, 787 ± 793.
[10] J. P. Kamerling, Pneumococcal Polysaccharides: A Chemical View, in:
Streptococcus pneumoniae (Ed.: A. Thomasz), Molecular Biology &
Mechanisms of Disease, New York, Mary Ann Liebert, 2000, pp. 81 ±
114.
[27] D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart, Org. Lett. 2000, 2,
701 ± 703.
[28] E. Meinjohanns, M. Meldal, T. Jensen, O. Werdelin, L. Galli-
Stampino, S. Mouritsen, K. Bock, J. Chem. Soc. Perkin Trans. 1 1997,
871 ± 884.
[29] L. F. Tietze, M. Arlt, M. Beller, K.-H. Glüsenkamp, E. Jähde, M. F.
Rajewski, Chem. Ber. 1991, 124, 1215 ± 1221.
[11] V. Pozsgay, Adv. Carbohydr. Chem. Biochem. 2000, 56, 153 ± 199.
[12] a) A. Ya. Chernyak, K. V. Antonov, N. K. Kochetkov, Bioorg. Khim.
1987, 13, 958 ± 966; b) A. Ya. Chernyak, K. V. Antonov, N. K.
Kochetkov, L. N. Padyukov, N. V. Tsvetkova, Carbohydr. Res. 1985,
141, 199 ± 212.
[13] P. J. Garegg, S. Oscarson, U. Tedebark, J. Carbohydr. Chem. 1998, 17,
587 ± 594.
[14] F. Suguwara, H. Nakayama, G. A. Strobel, T. Ogawa, Agric. Biol.
Chem. 1986, 50, 2251 ± 2259.
[30] L. F. Tietze, C. Schröter, S. Gabius, U. Brinck, A. Goerlach-Graw, H.-
J. Gabius, Bioconjugate Chem. 1991, 2, 148 ± 153.
[31] G. Giannini, R. Rappuoli, G. Ratti, Nucleic Acids Res. 1984, 12, 4063 ±
4069.
[32] a) D. M. Granoff, S. J. Holmes, M. T. Osterholm, J. E. McHugh, A. H.
Lucas, E. L. Anderson, R. B. Belshe, J. L. Jacobs, F. Medley, T. V.
Murphy, J. Infect. Dis. 1993, 168, 663 ± 671; b) D. C. Powers, E. L.
Anderson, K. Lottenbach, C. M. Mink, J. Infect. Dis. 1996, 173, 1014 ±
1018.
[15] F. A. Carey, K. O. Hodgson, Carbohydr. Res. 1970, 12, 463 ± 465.
[16] R. Verduyn, M. Douwes, P. A. M. van der Klein, E. M. Mösinger,
G. A. van der Marel, J. H. van Boom, Tetrahedron 1993, 49, 7301 ±
7316.
[17] P. J. Garegg, Pure Appl. Chem. 1984, 56, 845 ± 858.
[18] K. C. Nicolaou, N. Winssinger, J. Pastor, F. DeRoose, J. Am. Chem.
Soc. 1997, 119, 449 ± 450.
[19] A. H. Haines, Adv. Carbohydr. Chem. Biochem. 1976, 33, 11 ± 109.
[20] a) T. Ogawa, T. Kaburagi, Carbohydr. Res. 1982, 110, C12 ± C14;
b) H. P. Wessel, J. Carbohydr. Chem. 1988, 7, 263 ± 269.
[21] M. Bertolini, C. P. J. Glaudemans, Carbohydr. Res. 1970, 15, 263 ± 270.
[33] M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, F. Smith, Anal.
Chem. 1956, 28, 350 ± 356.
[34] P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H.
Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson,
D. C. Klenk, Anal. Biochem. 1985, 150, 76 ± 85.
[35] B. Benaissa-Trouw, D. J. Lefeber, J. P. Kamerling, J. F. G. Vliegenthart,
K. Kraaijeveld, H. Snippe, Infect. Immun. 2001, 69, 4698 ± 4701.
[36] J. H. Boyer, J. Hamer, J. Am. Chem. Soc. 1955, 77, 951 ± 954.
Received: April 12, 2001 [F3198]
Chem. Eur. J. 2001, 7, No. 20
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