Synthesis of Neisseria meningitidis serogroup W135 capsular oligosaccharides for immunogenicity comparison and vaccine development

Article abstract of DOI:10.1002/anie.201302540

Sweetening the deal: N.meningitidis serogroup W135 capsular oligosaccharides were synthesized in lengths from disaccharides to decasaccharides. Sera from mice immunized with these oligosaccharide-protein conjugates were examined by a glycan microarray (see picture) and bactericidal assay for antibody specificity and the ability to kill bacteria. Copyright

Full text of DOI:10.1002/anie.201302540

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Chemie
DOI: 10.1002/anie.201302540
Carbohydrate Vaccine
Synthesis of Neisseria meningitidis Serogroup W135 Capsular
Oligosaccharides for Immunogenicity Comparison and Vaccine
Development**
Chia-Hung Wang, Shiou-Ting Li, Tzu-Lung Lin, Yang-Yu Cheng, Tsung-Hsien Sun, Jin-
Town Wang, Ting-Jen R. Cheng, Kwok Kong Tony Mong, Chi-Huey Wong, and Chung-Yi Wu*
Neisseria meningitidis (meningococcus) is a Gram-negative
human pathogen that causes meningococcal diseases, such as
meningococcal meningitis and meningococcal septicemia.[1]
Based on the surface capsular oligosaccharides of the
organism, 13 serogroups of N. meningitidis have been iden-
tified, among which A, B, C, Y, and W135 are the major
pathogenic strains.[2] Serogroup A is the pathogen most often
implicated in the seasonal epidemic disease in the developing
countries of Asia and sub-Saharan Africa.[3] Serogroups B
and C cause the majority of cases in industrialized countries.[4]
Serogroups W135 and Y are responsible for the remaining
cases in developing countries.
The capsular polysaccharide plays an important role in
bacterial pathogenesis; its antiphagocytic properties help the
bacteria to escape from antibodies and complement deposi-
tion.[5] On the other hand, the unique structure of the capsular
polysaccharide also makes a good target for vaccine design.
The first polysaccharide vaccine was developed in
1974; however, owing to the poor immunogenicity of
polysaccharides, these vaccines typically elicit an IgM
response and fail to induce T-cell-dependent immun-
ity.[6] Moreover, polysaccharide vaccines are poor in
immunological memory, especially for children under
ride–protein conjugate vaccines were developed to improve
immunogenicity.[7] Currently, the major source of polysaccha-
rides for vaccine preparation is from acidic lysis of bacteria
and column chromatography purification.[8] Owing to limits of
purification, the obtained polysaccharide is heterogeneous
and therefore, vaccine quality is inconsistent. Hence, we were
interested in developing a synthesis of capsular polysaccha-
rides with defined lengths for a homogeneous vaccine.
Recently, we developed a chemical method to prepare pure
N. meningitidis serogroup C capsular polysaccharide, an a-
(2!9) oligosialic acid glycans of defined length.[9] Herein, we
focus on the synthesis of N. meningitidis serogroup W135
capsular oligosaccharide, which consists of a glycan repeating
unit of !6)-a-d-Galp-(1!4)-a-d-Neup5Ac(9OAc)-(2!
(Figure 1). To the best of our knowledge, the total synthesis
of this molecule has not been reported.
two-years old, who belong to the major risk group of
meningococcal diseases. Consequently, polysaccha-
Figure 1. Formula of N. meningitidis serogroup W135 capsular oligosaccharide
composed of a repeating unit of !6)-a-d-Galp-(1!4)-a-d-Neup5Ac(9OAc)-(2!.
[*] C.-H. Wang, S.-T. Li, Y.-Y. Cheng, Dr. T.-H. Sun, Dr. T.-J. R. Cheng,
Prof. C.-H. Wong, Prof. C.-Y. Wu
From a structural point of view, there are two major
challenges for such a synthesis: a-selectivity of the sialic acid
linkage and 1,2-cis galactoside formation. a-Sialylation is
challenging because of 1) the lack hydroxy group on the C-3
position for neighboring participation, 2) the strong electron-
withdrawing carboxylic group on the C-1 position, which
reduces the formation of the oxocarbenium, and 3) the
occurrence of a 2,3-elimination reaction during the glycosy-
lation reaction.[10] Many sialic acid donors designed to address
these problems have focused on three main characteristics:
leaving-group optimization, structure modification, or a com-
bination of both.[11] Moreover, the use of a nitrile solvent can
increase the a-selectivity.[12] Another synthetic challenge
comes from the 1,2-cis linkage for glyco-synthesis. Although
many approaches have been used to construct such a glycan
linkage, there is no general method to achieve this purpose.
The methods used to tackle this challenge include: 1) remote
electron-donating group participation;[13] 2) addition of
a bulky group at C-6 position for long-range participation;[14]
3) C-2 neighboring group participation.[15]
Genomics Research Center, Academia Sinica
128 Academia Road, Section 2, Nankang, Taipei, 115 (Taiwan)
E-mail: cyiwu@gate.sinica.edu.tw
C.-H. Wang, Prof. C.-H. Wong, Prof. C.-Y. Wu
Institute of Biochemistry and Molecular Biology
National Yang-Ming University
155, Linong Street, Section 2, Taipei, 112 (Taiwan)
T.-L. Lin, Prof. J.-T. Wang
Graduate Institute of Microbiology, National Taiwan University
College of Medicine
Taipei (Taiwan)
Prof. K. K. T. Mong
Department of Applied Chemistry, National Chiao-Tung University
Hsin-Chu (Taiwan)
[**] We thank the CDC of Taiwan for providing the W135 bacteria strain.
This work was supported by Academia Sinica and National Science
Council, Taiwan. (Grant no NSC 99-2113M-001-008-MY2 and NSC
101-2628M-001-004-MY2 to C.-Y.W.)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302540.
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Considering all these challenges, a strategy to synthe-
size the N. meningitidis serogroup W135 capsular oligo-
saccharide with various lengths is highly interesting. N.
meningitidis serogroup W135 capsular oligosaccharides
of defined lengths can be used to study the detailed
relationship between oligosaccharide length and immu-
nogenicity. After careful analysis of the structure of the
N. meningitidis serogroup W135 capsular oligosacchar-
ide, we decided to begin the synthesis from suitably
protected disaccharides 11 and 12 (Figure 2). These
disaccharides can be selectively opened at the N-acetyl-
5-N,4-O-carbonyl oxazolidinone ring under weakly basic
Scheme 1. Synthesis of disaccharide building blocks 11 and 12. Reagents
and conditions: a) TMSOTf, CH2Cl2, ꢀ708C, about 90%. TMSOTf=tri-
methylsilyl trifluoromethanesulfonate.
side and required a higher temperature for activation.[17b] In
this case, however, the b-phosphate sialoside 4 was fully
activated even at ꢀ708C with the galactoside acceptor 6. We
also obtained the a-isomer only product (12) in 91% yield
using 3 as a donor and 10 as an acceptor.
Oligosaccharide elongation was accomplished by way of
an iterative glycosylation and deprotection strategy, using
disaccharide 11 as a common donor for the [2+n] glycosyla-
tion reaction to construct up to decasaccharides. We used 12
as a reducing-end building block and selectively removed its
oxazolidinone ring using Zemplꢀn conditions to obtain 13 in
78% yield. Glycosylation of the disaccharide donor 11 and
alcohol acceptor 13 in the presence of NIS/TfOH activation in
CH2Cl2 at ꢀ408C for one hour gave the fully protected
tetrasaccharide 15 in 64% yield. Compound 15 was further
treated with Zemplꢀn conditions to open the oxazolidinone
ring and gave tetrasaccharide acceptor 16 in 75% yield. The
fully protected hexasaccharide 18 was synthesized by [2+4]
glycosylation using 11 and 16 with 52% yield as a single
stereoisomer. Repeating the oxazolidinone ring opening and
the same [2+n] glycosylation strategy, octasaccharide 21 and
decasaccharide 24 were synthesized. But with increasing
length of the oligosaccharide, the yields decreased to 46%
and 35%, respectively. Fortunately, these products were also
obtained as a single a isomer. The alcohol products of the
hexasaccharide 19, octasaccharide 22, and decasaccharide 25
were obtained from the fully protected oligosaccharide using
Zemplꢀn conditions in 70–80% yield (Scheme 2).
Finally, the completely deprotected compounds were
obtained by global deprotection of the alcohol compounds
13, 16, 19, 22, and 25. First, the TBDPS and isopropylidene
groups of the alcohol compounds were removed in the
presence of excess BF3·OEt2 at 08C for three hours owing to
the fluoride and acidic property of BF3. Second, the methyl
ester group was removed using a strong base, NaOH in
MeOH. Finally, all benzyl groups were removed by a hydro-
genation reaction with Pd(OH)2 catalyst and H2 in MeOH/
H2O. The final deprotected products 14, 17, 20, 23, and 26
were obtained in 45–60% yields over three steps (Scheme 2).
Notably, the estimated coupling constant for the anomeric
proton of galactose was about 3–4 Hz, and we confirmed that
only a-linked Gal-(1!4)-Neu5Ac oligosaccharides were
produced by the small value of this coupling constant (for
detailed synthetic procedures and compound characteriza-
tion, see the Supporting Information).
Figure 2. Retrosynthesis of N. meningitidis capsular oligosaccharide.
TBDPS=tert-butyldiphenylsilyl; STol=p-methylphenyl thio.
conditions[16] to give the 4-OH compound without affecting
the other protecting groups. The disaccharide STol leaving
group at the reducing end of the galactose residue was
designed for the next coupling reaction, and the azidopentane
linker at the reducing end was designed for immobilization
onto N-hydroxysuccinimide (NHS)-coated slides or conjuga-
tion to a carrier protein as vaccine candidates. Disaccharides
11 and 12 can be further disconnected into a suitably
protected sialyl phosphate donor 3 and thiol galactosides 6
and 10 owing to the concern of regio-deprotection or
stereoselective glycosylation. Compound 3 combined oxazo-
lidinone and the phosphate leaving group to increase the a-
selectivity[16,17] and was easily prepared from compound 1.[17a]
Detailed synthetic procedures for the monosaccharide build-
ing blocks 3, 6, 10 are shown in the Supporting Information.
We examined the conditions for reacting a- and b-
phosphate sialoside with galactoside. The a-phosphate sialo-
side 3 reacted with 6 under the activation of TMSOTf in
CH2Cl2 at ꢀ788C for ten minutes to give Neu5Ac-a-(2!6)-
Gal disaccharide 11 as a single isomer in 91% yield
(Scheme 1). The configuration of the disaccharide was
examined by NMR spectroscopy, and the newly formed a-
glycosidic bond was confirmed by a 3J(C1-H3ax) = 5.7 Hz
coupling constant. A previous report indicated that b-
phosphate sialoside was less reactive than a-phosphate sialo-
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fosuccinimidylpropionate) (DTSSP) to react with
the deprotected amine compounds 14, 17, 20, 23, and
26 in PBS buffer (pH 7.4) overnight. The disulfide
bond was then cleaved with dithiothreitol (DTT) at
408C for one hour to give the free thiol products 27,
28, 29, 30, and 31 as Michael donors in 70–75% yield.
To generate a reactive maleimide group on the
protein, diphtheria toxin (DT) mutant CRM197 was
reacted with N-(e-maleimidocaproyloxy)sulfosucci-
nimide ester (sulfo-EMCS) in PBS buffer (pH 8.0)
for one hour. The number of maleimide linkers on
the protein was determined by MALDI-TOF mass
spectrometry. On average, 20 maleimide linkers
were coupled to one CRM197 molecule. Oligosac-
charides were conjugated to the carrier protein
CRM197 by mixing thiol-modified oligosaccharides
27, 28, 29, 30, and 31 and maleimide-modified
CRM197 in PBS buffer (pH 7.4) for one hour
(Scheme 3) to obtain the glycoconjugates DT-2,
DT-4, DT-6, DT-8, and DT-10 with various carbohy-
drate epitopes on the DT. Again, the number of
oligosaccharides conjugated to the CRM197 was
determined by MALDI-TOF mass spectrometry
(Supporting Information, Table S1).
We examined the immunogenicity of these
synthetic glycoconjugates using
a mouse-serum
Scheme 2. Synthesis of oligosaccharides. Reagents and conditions: a) NaOMe,
MeOH, RT, 65–75%; b) NIS, TfOH, CH2Cl2, ꢀ408C, 64% for 15, 52% for 18, 46%
for 21, 35% for 24; c) BF3·OEt2, acetonitrile, 08C; NaOH, MeOH/H2O; Pd(OH)2/
H2, MeOH/H2O, 35–50%, over three steps. NIS=N-iodosuccinimide.
assay. In the experimental group, 6- to 8-week-old
female BALB/c mice (n = 5) were injected intra-
muscularly with conjugates containing 2 mg of oligo-
saccharide in 100 mL PBS buffer at two-week
intervals, and the antigens were formulated with
Many chemical approaches have been developed to
crosslink carbohydrates and proteins: 1) the Staudinger
ligation employs a substituted phosphite to react with the
azide-modified protein to form the carbohydrate–protein
conjugate through the formation of an amide bond;[18]
2) oxime conjugation introduces an aminooxy group on the
protein to react with an oligosaccharide containing an
aldehyde or ketone group;[19] 3) Michael addition often uses
thiol group addition to a maleimide to form a stable thioester
linkage;[20] and 4) copper (I)-catalyzed cycloaddition of azides
to alkynes (click chemistry) provides efficient glycoconjuga-
tion.[21] However, the triazole ring from the click chemistry
reaction produces an undesired immune response. Therefore,
we adopted the thiol–maleimide coupling method for carbo-
hydrate–protein conjugation because of its high efficiency in
sialic acid-rich compounds.[22] We began by using the com-
mercially available amine-reactive reagent 3,3-dithiobis(sul-
2 mg of an a-galactosylceramide derivative C34 or alum
adjuvant. PBS buffer alone was injected into the control
group of mice. Seven days after the third boost, blood samples
were collected for each mouse for serological immune
analysis by glycan microarray.
N. meningitidis W135 capsular di- to decasaccharides 14,
17, 20, 23, and 26, plus 70 other synthetic amine-containing
oligosaccharides (the formulas of these glycans are shown in
Table S2) were printed on NHS-coated glass slides. Details of
the microarray fabrication and detection procedure are
described in the Supporting Information. To investigate the
immunogenicity of different-length oligosaccharides, sera
collected from the mice were diluted 200-fold in PBST
buffer containing 3% BSA. The diluents were incubated with
the microarray at 48C for one hour to allow the induced
antibodies to bind to the oligosaccharides. Excess serum
antibodies were then washed out and the microarrays were
Scheme 3. Oligosaccharide conjugation to the carrier protein. Reagents and conditions: a) DTSSP, PBS buffer (pH 7.4), RT; DTT, 408C, 70–75%;
b) sulfo-EMCS, PBS buffer (pH 8.0), RT; c) PBS buffer (pH 7.4), RT.
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incubated with fluorescently labeled goat anti-mouse IgG
antibodies as the secondary antibody at 48C for one hour.
Finally, the slides were washed thoroughly and scanned at
635 nm wavelength with a microarray fluorescence chip
reader.
but the titer of IgM antibody was only 200 (Table S3).
Therefore, the use of a TD-antigen resulted in antibody
isotype switching from IgM to IgG.
We further looked into the distribution of the IgG
subclasses by incubating the oligosaccharide-coated micro-
array with secondary anti-mouse IgG1, IgG2a, IgG2b, IgG2c,
and IgG3 antibodies after the serum antibody binding. The
anti-IgG antibody in serum contained IgG1, IgG2b, IgG2c,
and IgG3 but no significant amount of IgG2a (Figure S4). We
observed that the IgG1 subclass was highest in the serum and
IgG3, a typical anti-carbohydrate antibody,[24] displayed
a high level in the serum.
A serum bactericidal assay (SBA) was used to demon-
strate the bactericidal abilities of the antibodies. Details of the
SBA are described in the Supporting Information.[25] Our
results showed that the bactericidal ability was roughly
correlated with the antibody level on the microarray. Accord-
ingly, serum from mice immunized with DT-2 showed no
bactericidal ability. The SBA titers in mice immunized with
DT-4 and DT-8 were 1/8 and 1/16 (Table 1). To summarize,
DT-2 does not induce antibodies with bactericidal abilities,
DT-4 was the minimum length required to induce bactericidal
antibodies, and DT-8 elicited the most abundant antibodies
with bactericidal effects.
In the group using C34 as an adjuvant, mice immunized
with DT-2 elicited antibody against the N. meningitidis
serogroup W135 capsular disaccharide 14, but did not cross
react with tetra- or longer oligosaccharides. This antibody also
recognized other similar disaccharides on the slide, including
Neu5Gc-a-(1!6)-Gal-a-(2! and Neu5Ac-a-(1!6)-Gal-b-
(2!. In contrast, antibodies induced by DT-4 bound tetra- to
decasaccharides 17, 20, 23, and 26, but did not recognize 14 or
other oligosaccharides on the slide (Figure S1). Antibodies
induced by DT-6, DT-8, and DT-10 also showed the same
pattern on the microarray as the DT-4 induced antibodies.
Therefore, we concluded that the antibodies induced by DT-4,
DT-6, DT-8, and DT-10 were very similar but different from
the antibodies induced by DT-2. Moreover, based on the
fluorescence intensity, DT-4, DT-6, DT-8, and DT-10 induced
antibodies bound to longer oligosaccharides with higher
affinity and DT-8 induced the most abundant antibody titers
(Figure 3).
In the group that used alum as an adjuvant, the antibodies
induced by DT-4, DT-6, DT-8, and DT-10 also differed from
DT-2 (Figure S2), and the patterns were overall very similar
Table 1: Serum bactericidal titers.[a]
Sera
Titer
DT-2/C34
DT-4/C34
DT-6/C34
DT-8/C34
DT-10/C34
n.d.[b]
1/8
1/8
1/16
1/4
[a] Bactericidal titer from sera of mice immunized with different length
oligosaccharide–protein conjugate. [b] No bactericidal activity.
In conclusion, we have synthesized Neisseria meningitidis
serogroup W135 capsular disaccharides to decasaccharides
with excellent stereoselectivity for each glycosylation step in
good yields. The highly a-stereoselective sialylation is due to
the combination of an N-acetyl-5-N,4-O-carbonyl protecting
group and a dibutyl phosphate as leaving group; while the
high a-stereoselectivity for galactosylation may be caused by
the long range participation of a bulky 6-O-group and
a benzyl group at the C-4 position. To overcome the intrinsi-
cally poor immunogenicity of oligosaccharides, these oligo-
saccharides were conjugated to a carrier protein (CRM197) in
the average number of 3–6, depending on the length of the
oligosaccharide. The carrier protein CRM197 provides pep-
tides to interact with MHC class II molecules on the antigen-
presenting cell (APC), followed by stimulation of the Th cell
for antibody maturation. Our results showed that induced
IgG antibody titers were much higher than IgM antibody
titers. Furthermore, analysis of the distribution of IgG
subclasses showed that the antigen predominantly elicited
IgG1 antibodies. Also, IgG3, a typical anti-carbohydrate
antibody, was found at high levels in the serum. Consequently,
the oligosaccharide–protein conjugate is a TD antigen, which
Figure 3. Comparison of the immunogenicity by microarray. Antibodies
elicited by DT-4–DT-10 conjugates had higher binding affinity with
longer oligosaccharides, and DT-8 recruited the highest quantity of
antibodies.
to the vaccines that used C34 as an adjuvant. However, the
titer of the antibodies induced using the alum adjuvant was
lower than using C34.
When the carbohydrate itself was used as an antigen, the
immune system produced
a thymus-independent (TI)
response, leading to predominantly IgM antibody. In contrast,
the carbohydrate–protein conjugates elicited a thymus-de-
pendent (TD) response,[23] and the ratio of IgG and IgM
changed. We used DT-8/C34-induced serum antibodies for
analysis of the antibody isotypes and subclasses. We first
determined DT-8 induced IgG and IgM antibody titers by
microarray by we defining the antibody titer as an s/N ratio of
fluorescence intensity lower than three. The results showed
that the anti-DT-8 IgG antibody titer was greater than 5 ꢁ 105,
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[10] G. J. Boons, A. V. Demchenko, Chem. Rev. 2000, 100, 4539 –
processes antibody isotype switching from IgM to IgG1.
Antibodies induced by DT-2 recognized only disaccharides,
but could not cross react with tetrasaccharides or longer. In
contrast, antibodies induced by DT-4, DT-6, DT-8, and DT-10
all recognized tetra- to decasaccharides but not disaccharides.
Patterns on the glycan microarray were the same for both
adjuvants, alum and C34. Therefore, we concluded that
antibodies induced by DT-4–DT-10 were similar, but different
from DT-2. Antibodies elicited by DT-2 showed no bacter-
icidal ability in the SBA experiment, while antibodies elicited
DT-4–DT-10 had good bactericidal ability. These results imply
that the minimum length of oligosaccharide required to make
effective antibodies is a tetrasaccharide, and further isotope-
labeling experiments are ongoing. Furthermore, with glycans
in a single and homogeneous form, we can control the number
of glycans on the protein to modulate the immunogenicity.
Thus, we have demonstrated that synthetic tetrasaccharide–
protein conjugates are a promising candidate for producing
anti-Neisseria meningitidis serogroup W135 vaccines.
4566.
[11] a) M. J. Kiefel, M. von Itzstein, Chem. Rev. 2002, 102, 471 – 490;
b) X. Chen, A. Varki, ACS Chem. Biol. 2010, 5, 163 – 176;
c) D. K. Ress, R. J. Linhardt, Curr. Org. Synth. 2004, 1, 31 – 46;
d) R. L. Halcomb, M. D. Chappell, J. Carbohydr. Chem. 2002, 21,
723 – 768; e) C. H. Hsu, S. C. Hung, C. Y. Wu, C. H. Wong,
Angew. Chem. 2011, 123, 12076 – 12129; Angew. Chem. Int. Ed.
2011, 50, 11872 – 11923; f) H. Ando, M. Kiso in Glycoscience
Chemistry and Chemical Biology (Eds.: B. O. Fraser-Reid, K.
Tatsuta, J. Thiem), Springer, Berlin, 2008, pp. 1313 – 1360.
[12] a) R. R. Schmidt, M. Behrendt, A. Toepfer, Synlett 1990, 694 –
696; b) H. Lçnn, K. Stenvall, Tetrahedron Lett. 1992, 33, 115 –
116.
´
[13] M. Miljkovic, D. Yeagley, P. Deslongchamps, Y. L. Dory, J. Org.
Chem. 1997, 62, 7597 – 7604.
[14] a) K. Fukase, Y. Nakai, T. Kanoh, S. Kusumoto, Synlett 1998, 84 –
86; b) K. Fukase, Y. Nakai, K. Egusa, J. A. Porco, S. Kusumoto,
Synlett 1999, 1074 – 1078; c) H. Tokimoto, Y. Fujimoto, K.
Fukase, S. Kusumoto, Tetrahedron: Asymmetry 2005, 16, 441 –
447; d) L. Q. Chen, F. Z. Kong, Tetrahedron Lett. 2003, 44, 3691 –
3695; e) A. Imamura, H. Ando, S. Korogi, G. Tanabe, O.
Muraoka, H. Ishida, M. Kiso, Tetrahedron Lett. 2003, 44, 6725 –
6728.
[15] a) J. H. Kim, H. Yang, J. Park, G. J. Boons, J. Am. Chem. Soc.
2005, 127, 12090 – 12097; b) D. J. Cox, A. J. Fairbanks, Tetrahe-
dron: Asymmetry 2009, 20, 773 – 780; c) E. A. Mensah, F. Yu,
H. M. Nguyen, J. Am. Chem. Soc. 2010, 132, 14288 – 14302.
[16] D. Crich, W. Li, J. Org. Chem. 2007, 72, 2387 – 2391.
[17] a) H. Tanaka, Y. Nishiura, T. Takahashi, J. Am. Chem. Soc. 2006,
128, 7124 – 7125; b) C. H. Hsu, K. C. Chu, Y. S. Lin, J. L. Han,
Y. S. Peng, C. T. Ren, C. Y. Wu, C. H. Wong, Chem. Eur. J. 2010,
16, 1754 – 1760.
[18] C. Grandjean, A. Boutonnier, C. Guerreiro, J. M. Fournier, L. A.
Mulard, J. Org. Chem. 2005, 70, 7123 – 7132.
[19] J. Kubler-Kielb, V. Pozsgay, J. Org. Chem. 2005, 70, 6987 – 6990.
[20] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura,
Y. C. Lee, Biomacromolecules 2005, 6, 880 – 884.
[21] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001,
113, 2056 – 2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021;
Angew. Chem. 2001, 113, 2056 – 2075; b) S. Hotha, S. Kashyap, J.
Org. Chem. 2006, 71, 364 – 367.
[22] Y. L. Huang, J. T. Hung, S. K. Cheung, H. Y. Lee, K. C. Chu, S. T.
Li, Y. C. Lin, C. T. Ren, T. J. Cheng, T. L. Hsu, A. L. Yu, C. Y.
Wu, C. H. Wong, Proc. Natl. Acad. Sci. USA 2013, 110, 2517 –
2522.
Received: March 26, 2013
Revised: June 11, 2013
Published online: July 10, 2013
Keywords: bactericidal · N. meningitidis · oligosaccharides ·
.
total synthesis · vaccine
[1] a) Y. L. Tzeng, D. S. Stephens, Microbes Infect. 2000, 2, 687 –
700; b) M. Virji, Nat. Rev. Microbiol. 2009, 7, 274 – 286.
[2] a) N. E. Rosenstein, B. A. Perkins, D. S. Stephens, T. Popovic,
J. M. Hughes, N. Engl. J. Med. 2001, 344, 1378 – 1388; b) D. S.
Stephens, FEMS Microbiol. Rev. 2007, 31, 3 – 14.
[3] M. Achtman, Trends Microbiol. 1995, 3, 186 – 192.
[4] D. S. Stephens, B. Greenwood, P. Brandtzaeg, Lancet 2007, 369,
2196 – 2210.
[5] a) M. R. Spinosa, C. Progida, A. Tala, L. Cogli, P. Alifano, C.
Bucci, Infect. Immun. 2007, 75, 3594 – 3603; b) M. C. Schneider,
R. M. Exley, S. Ram, R. B. Sim, C. M. Tang, Trends Microbiol.
2007, 15, 233 – 240.
[6] a) A. Gonzꢂlez-Fernꢂndez, J. Faro, C. Fernꢂndez, Vaccine 2008,
26, 292 – 300; b) A. Weintraub, Carbohydr. Res. 2003, 338, 2539 –
2547.
[7] a) M. D. Snape, A. J. Pollard, Lancet Infect. Dis. 2005, 5, 21 – 30;
b) G. Ada, D. Isaacs, Clin. Microbiol. Infect. 2003, 9, 79 – 85;
c) M. P. Girard, M. P. Preziosi, M. T. Aguado, M. P. Kieny,
Vaccine 2006, 24, 4692 – 4700.
[8] A. Bardotti, G. Averani, F. Berti, S. Berti, C. Galli, S. Giannini, B.
Fabbri, D. Proietti, N. Ravenscroft, S. Ricci, Vaccine 2005, 23,
1887 – 1899.
[23] a) K. E. Stein, D. A. Zopf, B. M. Johnson, C. B. Miller, W. E.
Paul, J. Immunol. 1982, 128, 1350 – 1354; b) K. E. Stein, J. Infect.
Dis. 1992, 165 Suppl 1, S49 – 52.
[24] R. M. Perlmutter, D. Hansburg, D. E. Briles, R. A. Nicolotti,
J. M. Davie, J. Immunol. 1978, 121, 566 – 572.
[25] a) S. Romero-Steiner, J. Fernandez, C. Biltoft, M. E. Wohl, J.
Sanchez, J. Feris, S. Balter, O. S. Levine, G. M. Carlone, Clin.
Diagn. Lab. Immunol. 2001, 8, 1115 – 1119; b) Y. Schlesinger,
D. M. Granoff, J. Am. Med. Assoc. 1992, 267, 1489 – 1494.
[9] K. C. Chu, C. T. Ren, C. P. Lu, C. H. Hsu, T. H. Sun, J. L. Han, B.
Pal, T. A. Chao, Y. F. Lin, S. H. Wu, C. H. Wong, C. Y. Wu,
Angew. Chem. 2011, 123, 9563 – 9567; Angew. Chem. Int. Ed.
2011, 50, 9391 – 9395.
Angew. Chem. Int. Ed. 2013, 52, 9157 –9161
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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