Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen Containing a dd-Heptoglycan

Article abstract of DOI:10.1002/anie.202004267

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo-polysaccharide (LPS) O-antigens that contain Lewis determinant oligosaccharides and unique dd-heptoglycans has not yet been ident

Full text of DOI:10.1002/anie.202004267

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Accepted Article
Title: Chemical Synthesis and Immunological Evaluation of
Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen
Containing a Unique DD-Heptoglycan
Authors: Jian Yin, guangzong Tian, Jing Hu, Chunjun Qin, Lingxin Li,
Xiaopeng Zou, Juntao Cai, and Peter H. Seeberger
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10.1002/anie.202004267
Angewandte Chemie International Edition
RESEARCH ARTICLE
Chemical Synthesis and Immunological Evaluation of
Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen
Containing a Unique DD-Heptoglycan
Guangzong Tian,[a][b]+ Jing Hu,[c]+ Chunjun Qin,[a]+ Lingxin Li,[a] Xiaopeng Zou,[a][b] Juntao Cai,[a][b] Peter
H. Seeberger,[b] and Jian Yin*[a]
Dedicated to the 100th anniversary of the birth of Prof. Dr. Raymond Urgel Lemieux
[a]
G. Tian, C. Qin, L. Li, X. Zou, J. Cai, Prof. J. Yin
Key Laboratory of Carbohydrate Chemistry and Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University
Lihu Avenue 1800, Wuxi, Jiangsu Province, 214122, P.R. China
E-mail: jianyin@jiangnan.edu.cn
[b]
G. Tian, X. Zou, J. Cai, Prof. P. H. Seeberger
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mühlenberg 1, Potsdam, 14476, Germany
J. Hu
Wuxi School of Medicine, Jiangnan University
Lihu Avenue 1800, Wuxi, Jiangsu Province, 214122, P.R. China
These authors contributed equally
[c]
+
Abstract: The development of glycoconjugate vaccines against
Helicobacter pylori is challenging. An exact epitope of the H. pylori
lipopolysaccharides (LPS) O-antigens that contain Lewis
determinant oligosaccharides and unique DD-heptoglycans has not
yet been identified. Here, we report the first total synthesis of H.
pylori serotype O6 tridecasaccharide O-antigen containing a terminal
Ley tetrasaccharide, a unique α-(1→3)-, α-(1→6)- and α-(1→2)-
linked heptoglycan, and a β-D-galactose connector through an [(2 ×
1) + (3 + 8)] assembly sequence, which may serve as reference for
the syntheses of other sterically hindered long-chain glycans. Seven
oligosaccharides covering different portions of the entire O-antigen
were prepared for immunological investigations with a particular
focus on elucidation of the roles of DD-heptoglycan and Ley
tetrasaccharide. Glycan microarrays analysis of sera from rabbits
immunized with isolated serotype O6 LPS revealed a humoral
immune response to the α-(1→3)-linked heptoglycan, which is the
key motif for designing glycoconjugate vaccines for H. pylori
serotype O6.
Lipopolysaccharide (LPS) O-antigens, a major cell surface
component of H. pylori, have been recognized as attractive
vaccine candidates.[15,16] The structures of LPS O-antigens from
six known H. pylori serotypes (O1 to O6) and various untypable
H. pylori strains have been reported.[17-19] A series of unique DD-
heptoglycans consisting of seven to thirteen D-glycero-α-D-
manno-heptopyranoses (DD-heptoses) and three different
glycosidic linkages α-(1→2), α-(1→3) and α-(1→6) (Figure 1)
has been found in H. pylori serotype O3,[20] O5,[16] O6,[20] strain
MO19[21] and a number of untypable H. pylori strains isolated
from asymptomatic hosts.[18] Until now, other glycans comprising
solely DD-heptose was only found in the LPS outer core region of
Klebsiella pneumoniae strain 01/R20 that has been elucidated
as an α-(1→2)-linked DD-heptoglycan.[22] Most H. pylori LPS O-
antigens (O1, O3, O4, O5 and O6) are terminated by
oligosaccharides resembling mammalian histo-blood-group
antigens, such as Lewis determinants (Lea, Leb, sialyl Lex, Lex
and Ley).[17] These mammalian histo-blood-group antigen
analogues have not been found in untypable H. pylori strains
from asymptomatic hosts.[18,19] Lower pathogenicity of H. pylori
strains lacking Lewis determinant oligosaccharides suggests the
notion that the extended heptoglycan region camouflage H. pylo-
Introduction
Helicobacter pylori infects half of the world’s population,[1] and is
strongly associated with chronic gastritis, peptic ulcer and
gastric cancer.[2-4] Infections with this bacterium increase the risk
for developing gastric cancer two- to eightfold,[5–8] and led to its
classification as a class I carcinogen.[9] Combined antimicrobial
and anti-acid therapy can eradicate H. pylori infections,[10] but
the overuse of currently recommended treatments has resulted
in antimicrobial resistance.[11] Antimicrobial cure of H. pylori
infections does not prevent re-infection, such that vaccines are
urgently needed.[12,13] Despite significant efforts and a fusion
protein-based vaccine consisting of urease B subunit and heat-
labile enterotoxin B subunit licensed in China since 2009,[14] no
H. pylori vaccine is marketed globally today.
Figure 1. Structure of O-antigen of H. pylori serotype O6 LPS.
1
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10.1002/anie.202004267
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RESEARCH ARTICLE
Figure 2. Retrosynthetic analysis of O-antigen tridecasaccharide of H. pylori serotype O6.
ri for immune escape.[17,18] On the other hand, several studies
suggested the existence of specific epitopes sharing a common
structural motif located in H. pylori O-antigens, since patients’
sera can recognize immunogenic LPS region, rather than Lewis
antigen analogues.[23,24] The roles of Lewis determinant
oligosaccharides and heptoglycans in the H. pylori-immune
system interactions remain controversial due to lack of
molecular understanding. Structurally-defined carbohydrate
antigens are required to establish the roles of these two
oligosaccharide portions of H. pylori O-antigens.[25] Chemical
synthesis offers an attractive means to procure the specific
carbohydrate antigens needed to elucidate their roles in H. pylori
pathogenicity and advance the development of glycoconjugate
vaccines.[26] Various Lewis determinant oligosaccharides have
been chemically[27] and enzymatically[28] synthesized. The DD-
heptoglycan has not yet been synthesized. The O-antigen of H.
pylori serotype O6, that is divided into two subtypes O6A and
O6B,[20] is a representative glycan containing both Lewis
determinant oligosaccharides and DD-heptoglycan. The H. pylori
O6A O-antigen tridecasaccharide is composed of a terminal Ley
tetrasaccharide and a DD-heptoglycan consisting of eight DD-
heptoses (two α-(1→2)-, four α-(1→3)- and one α-(1→6)-
glycosidic linkages), that are connected via a β-D-galactose
residue (Figure 1).[20] H. pylori serotype O6B expresses a
truncated O-antigen consisting solely of the DD-heptoglycan
(Figure 1).[20] The chemical synthesis of the H. pylori O6A O-
antigen tridecasaccharide and O6B O-antigen octasaccharide is
essential for immunological identification of H. pylori-expressing
Ley tetrasaccharide and DD-heptoglycan. The chemical synthesis
of H. pylori O6 O-antigen tridecasaccharide poses a number of
challenges, especially the steric hindrance of DD-heptoglycan
that influences glycosylation efficiencies. To improve the yield of
glycosylation reactions, an electronic effect-based protecting
group strategy,[29,30] optimization of leaving group/activator
condition[31] and steric effect-based assembly sequence
planning[32-34] served well to tune the reactivities of the glycosyl
donor and acceptor. Here, we report the first total synthesis of H.
pylori serotype O6A and O6B O-antigens by a trial-and-error
approach based on the reactivities of the glycosyl donor and
acceptor, to prepare sterically hindered long-chain glycans.
Glycan array screening identified the key epitopes that can be
recognized by IgG antibodies against native LPS as a basis for
the design of synthetic carbohydrate conjugate vaccines.[35]
Therefore,
a library of synthetic serotype O6 O-antigen
fragments has been established for immunological investigations,
particularly to elucidate the antigenicity of the Ley
oligosaccharide and DD-heptoglycan.
Results and Discussion
Retrosynthetic Analysis. An aminopropyl linker was placed at
the reducing end of synthetic tridecasaccharide
1
for
immobilization on glycan array surfaces or conjugation to carrier
proteins (Figure 2). The assembly of long-chain glycan 1 relies
on the convergent assembly of two judiciously selected
oligosaccharide fragments. Considering the steric hindrance of
DD-heptoglycan, the final assembly point should not reside within
the DD-heptoglycan part. Thus, retrosynthetic analysis initiated
from a [5 + 8] coupling of a pentasaccharide 3 and a hepto-
octasaccharide 2. For the assembly of octasaccharide 2, a [3 +
(2 + 3)] coupling from the reducing to the nonreducing end was
adopted, but not a [(3 + 2) + 3] coupling from the nonreducing to
2
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Scheme 1. Diversity-oriented synthesis of heptosides 9, 10 and 11.
the reducing end mainly due to worries over the low reactivity of
the large glycosyl donor. Consideration of the orthogonal
protecting groups were required for regioselective glycosylation,
three building blocks 9, 10 and 11 were identified for the
assembly of heptoglycans. Making use of previous syntheses of
Ley tetrasaccharide,[27] pentasaccharide 3 was designed to be
prepared via coupling of Ley tetrasaccharide donor 7 and thio-
galactoside 8. A [(2 × 1) + 2] coupling strategy was chosen to
assemble Ley-tetrasaccharide from three building blocks 12, 13
and 14.
Synthesis of Manno-Heptosides 9, 10 and 11. A diversity-
oriented synthesis of manno-heptosides 9, 10 and 11 started
with thioglycoside 15[36] (Scheme 1). Key heptoside 16 bearing
two free hydroxyl groups was obtained from 15 in an overall
yield of 44%. Treatment of 16 with benzyl bromide (BnBr) and
sodium hydride (NaH) in dimethylformamide (DMF) afforded
benzylated thioglycoside 17. The isopropylidene group in 17 was
smoothly removed under acidic condition to produce diol 18 that
was employed for the divergent synthesis of manno-heptosides
9 and 11. An O2 regioselective benzoylation of 18 via an
orthoester intermediate afforded 19 in 80% overall yield. Next,
treatment with levulinic acid and dicyclohexylcarbodiimide (DCC)
converted 19 to fully protected thioglycoside 20 in 82% yield.
The hydrolyzed product of 20 was transformed to Schmidt donor
9 that can be glycosylated with thioglycoside acceptors. The tin-
mediated regioselective benzylation at O3 of the diol 18
furnished 21 that was converted to the O2 acetylated
thioglycoside 11 in 72% overall yield. To synthesize the third
manno-heptoside 10, regioselective O7 benzylation of diol 16
produced 7-OBn product 22 in 66% yield. The O6 protection of
compound 22 by using levulinic acid and DCC gave 6-O-Lev
derivative 23 quantitatively. Subsequent removal of the
isopropylidene group and acetylation furnished 24, which was
transformed to Schmidt donor 10 in good overall yield.
Synthesis of Heptoglycan-Octasaccharide 2. With three
manno-heptosides 9, 10 and 11 in hand, the assembly of octa-
heptan 2 started from the reducing to the non-reducing end
(Scheme 2). As the first step, a propylamine linker was
introduced to the anomeric position of thioglycoside 11 to afford
25 in 85% yield as the sole isomer. After removal of the O2
acetyl group, desired glycosyl acceptor 26 was coupled with
thioglycoside 11 to produce 1→2-linked di-heptan 27 in 76%
yield with exclusive α-selectivity. Cleavage of an acetyl group
converted 27 to alcohol 28 that was reacted with thioglycoside
24 to give 1→2-linked tri-heptan 29 along with inseparable
byproducts. Glycosylation of 28 with Schmidt donor 10 promoted
by trimethylsilyl trifluoromethanesulfonate (TMSOTf) smoothly
furnished tri-heptan 29 in 66% yield with exclusive α-selectivity.
Next, to enhance the glycosylation efficiencies of the penta-
heptan containing four 1→3-linked heptosidic bonds, manno-
heptoside 9 was used to prepare two donors: dimer 5 and trimer
6 (for detailed syntheses please see Supporting Information). A
subsequent [3 + (2 + 3)] coupling starting from the delevulinated
tri-heptan 4, rather than a [(3 + 2) + 3] coupling, provided an
easy entry to the assembly of octa-heptan 2. Thus, the tri-heptan
4 was smoothly coupled with dimer 5 to afford penta-heptan 30
containing 1→6-linked heptosidic bond. After removal of levulinyl
group, acceptor 31 was glycosylated with trimer 6 under NIS and
TMSOTf promotion at -10 °C to produce octa-heptan 32 in good
yield and complete α-selectivity. The further delevulination gave
the desired glycosyl acceptor 2 in 90% yield.
Synthesis of Ley-Containing Pentasaccharide. The synthesis
of the Ley tetrasaccharide started from the coupling of building
block 14 (see Supporting Information) and trifluoroacetimidate
glycosyl donor 13 (see Supporting Information) catalyzed by
TMSOTf, producing disaccharide 33 in 63% yield with an α/β
ratio of 1:9 (Scheme 3). Cleavage of the benzoyl and levulinyl
groups in 33 with sodium methoxide treatment afforded a
phthalimide ring-opened diol intermediate, which was refluxed in
3
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Scheme 2. Synthesis of heptoglycan-octasaccharide 2.
pyridine and 10% aqueous AcOH to give acceptor 34 in 86%
overall yield. Condensation of disaccharide 34 with L-fucosyl
donor 12[37] under the TMSOTf activation in a mixture of Et2O
and CH2Cl2 at -40 °C gave Ley tetrasaccharide 35 in 74% yield
and complete α-selectivity. Thioglycoside 35 was transformed to
the corresponding N-phenyl trifluoroacetimidate glycosyl donor 7
that can be glycosylated with thioglycoside acceptors. Synthesis
of the pentasaccharide using tetrasaccharide donor 7 and thiogl-
ycoside acceptor 8 (see Supporting Information) failed, but gave
mainly a by-product identified as 35. Likely, aglycon transfer
occurred under the TMSOTf-catalyzed glycosylation conditions
due to unmatched reactivities between the glycosyl donor and
acceptor.[38]
To address the problem of aglycon transfer, thioglycoside 8
was transformed to the O1-TBS compound 36 (see Supporting
Information) (Scheme 4). Unfortunately, the glycosylation of
thioglycoside 35 and acceptor 36 afforded pentasaccharide 37 in
low yield (13%). D-Glucosamine derivatives show different
glycosyl donor reactivities depending on the protecting group pa-
Scheme 4. Synthesis of Ley-containing pentasaccharide 40.
Scheme 3. Attempt to Ley-containing pentasaccharide.
4
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Scheme 5. Attempted assembly sequence for the synthesis of serotype O6 O-antigen tridecasaccharide.
tterns of 2-NH2: N-2,2,2-trichlorethoxycarbonyl-(Troc)
>
N-
and donor degradation products suggested that the failure of the
glycosylation reaction may be due to steric hindrance in
octasaccharide acceptor 2. In view of the successful coupling of
tetrasaccharide donor 38 and D-galactose acceptor 36,
glycosylation of D-galactose donor 42 and octasaccharide 2
furnished a nonasaccharide, which can be transformed to a D-
galactoside terminated acceptor (Scheme 5B) (see Supporting
Information). A [4 + 9] coupling of nonasaccharide acceptor with
both tetrasaccharide donor 38 and 41 failed to produce
tridecasaccharide. Decomposition of the donors was detected
during these reactions. The reactivity of the C3 hydroxyl group in
octasaccharide 2 may be dependent on the steric effect as it
reacts with monosaccharide donor 42, but not pentasaccharide
donor 40. The efficient union of tetrasaccharide 38 and
monosaccharide acceptor 36, and the failure of the glycosylation
between tetrasaccharide 38 and nonasaccharide acceptor,
indicate that the reactivity of the C3 hydroxyl group in D-
galactoside, is influenced by the patterns at the reducing end.
A [(4 + (1 + 3)) + 5] assembly sequence was adopted to
tune the reactivity of the C3 hydroxyl group in D-galactoside by
placing a trisaccharide at the reducing end (Scheme 5C) (see
phthalimido-(Phth) > azido- > N-acetyl-.[29] Since the relative
reactivity value (RRV) (28.6) of 2-NHTroc-D-glucosamine donor
is higher than that (1.0 - 3.5) of the 2-NPhth-D-glucosamine
donor,[29] Troc was chosen as an alternative protecting group for
the C2 amino group of Ley tetrasaccharide. Selective removal of
the Phth group using ethylene diamine in n-BuOH at an elevated
temperature (95 °C)[39] and subsequent treatment with TrocCl in
pyridine furnished the tetrasaccharide 38. Glycosylation of donor
38 and acceptor 36 under TfOH and NIS activation at -20 °C
afforded the β-linked product 39 in 70% yield. Partial cleavage of
Troc under TBAF conditions forced us to choose HF-pyridine to
remove anomeric TBS group. The obtained hemiacetal was
transformed to the corresponding trifluoroacetimidate 40 in 42%
overall yield.
Synthesis of H. pylori O6 O-Antigen Tridecasaccharide 1
and Related Oligosaccharides. The tridecasaccharide was
assembled via a [5 + 8] coupling (Scheme 5A). Unfortunately,
the glycosylation of octasaccharide acceptor
2
and
pentasaccharide donor 40 in the presence of TMSOTf at 0 °C
failed to give the target tridecasaccharide. Unreacted acceptor
5
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RESEARCH ARTICLE
Scheme 6. Synthesis of serotype O6 O-antigen tridecasaccharide 1 by [(2 × 1) + (3 + 8)] strategy.
Supporting Information). The condensation of 42 with
trisaccharide 43 catalyzed by TMSOTf produced the
corresponding tetrasaccharide in 68% yield as β-anomer, before
removal of the levulinyl group afforded the corresponding
acceptor. Union of donor 41 and the tetrasaccharide acceptor
under TMSOTf activation gave the β-linked octasaccharide in
51% yield, indicating that the acceptor activity of 3-OH in D-
galactoside bearing tri-heptan unit at the reducing end is suitable
for the Ley tetrasaccharide donor. However, the [8 + 5] coupling
of octasaccharide donor and penta-heptan acceptor 31 failed to
produce tridecasaccharide, confirming the idea that the final
assembly point may not be chosen inside the sterically hindered
DD-heptoglycan part. Hence, an evaluation of the reactivity of the
C3 hydroxyl group in D-galactose terminated nonasaccharide
acceptor was needed. An assembly sequence of [1 + (1 + 8)]
was adopted and failed in the glycosylation of the
nonasaccharide acceptor and the D-glucosamine donor 44[40]
(Scheme 5D) (see Supporting Information), showing that the
octo-heptan fragment reduced the reactivity of the C3 hydroxyl
group in D-galactoside too drastically. Octo-heptan 2 should be
synthesized initially while the glycosylation at 3-OH of D-
galactoside has to be achieved at the D-galactose
monosaccharide stage.
Next, a [(2 × 1) + (3 + 8)] assembly sequence based on
trisaccharide donor 45 was adopted, where the glycosylation of
trisaccharide 45 and octasaccharide 2 was followed by L-
fucosylation (Scheme 6). Condensation of donor 46 (see
Supporting Information) and acceptor 47[40] was catalyzed by
TMSOTf and produced disaccharide 48 in 53% yield as β-
anomer. The glycosylation of 48 with acceptor 36 under TfOH
and NIS activation at -20 °C afforded the β-linked trisaccharide
49 in 70% yield. Removal of the TBS by TBAF gave hemiacetal
that was transformed to the corresponding N-phenyl
trifluoroacetimidate 45 in 87% overall yield. A replacement of N-
6
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Figure 3. A glycan library related to the H. pylori O6 O-antigen.
Troc by N-trichloroacetyl (TCA) group avoided the cleavage of
amino protecting group in the presence of TBAF. The union of
N-TCA group in 52 by treatment with Zn-AcOH furnished the
corresponding NHAc derivative, while all ester groups were
removed by NaOH and NaOMe in a mixture of methanol and
tetrahydrofuran. Final deprotection of all benzyl ether and benzyl
carbamate groups with Pd/C hydrogenation gave target
tridecasaccharide 1 in 82% overall yield. The NMR spectrum of
compound 1 is identical with that described in the reference (see
Supporting Information).[20]
45 and octasaccharide
2 catalyzed by TMSOTf led to
undecasaccharide 50 in 65% yield as β-anomer.
Undecasaccharide acceptor 51 was obtained through cleavage
of the Lev ester by treatment with hydrazine acetate. A
glycosylation reaction between L-fucoside 12 (4 eq) and
undecasaccharide acceptor 51 in the presence of TMSOTf gave
tridecasaccharide 52 in 52% yield and complete α-selectivity.
The failed glycosylation of pentasaccharide donor 40 and
octasaccharide acceptor 2, the success of D-galactose donor 42
and trisaccharide donor 45 to glycosylate 2, implies that the
acceptor reactivity of the octasaccharide 2 may be dependent on
the steric effect. On the other hand, the decomposition of L-
fucoside-containing donors 40, 38 and 41 indicated that the
failure of [5 + 8] and [4 + (1 + 8)] coupling may be attributed to
the lability of the fucosyl linkages during the acid-mediated
glycosylation reactions with long-chain glycans. Reduction of the
Finally, based on the Ley tetrasaccharide unit, DD-
heptoglycan unit and different glycosidic linkages, six
oligosaccharides (53−58) related to the H. pylori O6 O-antigen
were designed and chemically synthesized (Figure 3) (see
Supporting Information). Sufficient amounts of pure and well-
defined glycans for the generation of glycan microarrays and
glycoconjugates were obtained.
Antigenicity Evaluation of Synthetic H. pylori O6 O-Antigen.
The synthetic oligosaccharides were printed on microarray
slides via the reducing end aminopropyl linker (Figure 4) to ass-
7
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10.1002/anie.202004267
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RESEARCH ARTICLE
the tridecasaccharide, was significantly influenced by the
functional patterns at the reducing end. Seven oligosaccharides
decorated with
a propylamine linker laid the basis for
immunological investigations into the role of the unique and
conserved heptoglycan. Polyclonal rabbit anti-serotype O6
serum from animals immunized with isolated serotype O6 LPS
contains antibodies that very specifically bind α-(1→3)-linked
heptoglycans 55, 56 and 58 but not any other sequences. The α-
(1→3)-linked heptoglycans are antigenic epitopes while the Ley
tetrasaccharide may rather serve as a decoy to the mammalian
immune system, which makes H. pylori like a wolf in sheep’s
clothing[19]. The α-(1→3)-linked heptoglycan is a key motif for
designing glycoconjugate vaccines for H. pylori serotype O6.
Synthetic oligosaccharides are key for the identification of
defined cell-surface glycan epitopes as a basis for vaccine
development. The challenging synthesis of tridecasaccharide 1
based on trial-and-error approach taught valuable lessons for
the syntheses of other sterically hindered long-chain glycans.
Acknowledgements
Figure 4. Evaluation of synthetic oligosaccharide fragments by glycan
microarray. (a) Representative array scan of H. pylori O6 LPS immunized
rabbit sera (Left) and printing pattern (Right). (b) Quantification of mean
fluorescence intensity (MFI) values of oligosaccharide fragments. Error bars
represent SEM of two spots of two separate arrays.
We are grateful for National Natural Science Foundation of
China (21877052, 21907039), Natural Science Foundation of
Jiangsu Province (BK20180030, BK20190575), National First-
class Discipline Program of Light Industry Technology and
Engineering (LITE2018-14), the Max Planck Society
International Partner Group Program and China Scholarship
Council (CSC). P.H.S. thanks the Max-Planck Society for
generous financial support.
ess the antigenicity of serotype O6 Ley tetrasaccharide and DD-
heptoglycans. Glycan arrays containing all oligosaccharide
fragments were screened with the antisera of rabbits after
immunization with purified H. pylori serotype O6 LPS. The
purified serotype O6 LPS was also printed onto the glass slides
as positive control.
Conflict of interest
Among the synthetic oligosaccharides, rabbit IgG antibodies
bound specifically to α-(1→3)-linked heptoglycans 55, 56 and 58
but not to any of the other sequences, including α-(1→2)-,
(1→6)-linked heptosides and Ley tetrasaccharide. Moreover, 1
capped with Ley tetrasaccharide was not recognized by the
rabbit antisera, compared with 58. The mammalian histo-blood-
group antigen Ley tetrasaccharide in the H. pylori O6 O-antigen
can’t be recognized by antisera but also impede the IgG
recognition. Oligosaccharides 56 and 58 that contain more α-
(1→3)-linked heptoside motifs bound to rabbit IgG antibodies
with a little higher intensity than 55 that contains only one α-
(1→3)-linked heptoside motif. The α-(1→3)-linked heptoglycan is
a key epitope for designing glycoconjugate vaccines for H. pylori
serotype O6.
The authors declare no conflict of interest.
Keywords: Helicobacter pylori • immunology • O-antigen • total
synthesis • tridecasaccharide
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Conclusion
The first total synthesis of H. pylori serotype O6 O-antigen
tridecasaccharide 1 and related substructures 53-58 provided
access to pure glycans not available by isolation from natural
sources. A trial-and-error approach based on five elaborate
assembly sequences was employed and provided an efficient [(2
× 1) + (3 + 8)] assembly of tridecasaccharide 1. The steric
hindrance of DD-heptoglycan, necessitated the final assembly
point to be outside the DD-heptoglycan part. The reactivity of the
C3 hydroxyl group in D-galactoside, the key assembly point of
[9]
[10] S. M. Campo, A. Zullo, C. Hassan, S. Morini, Recent Pat. Antiinfect
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Entry for the Table of Contents
The Helicobacter pylori serotype O6 tridecasaccharide O-antigen containing a terminal Ley tetrasaccharide and a unique DD-
heptoglycan was chemically synthesized for the first time. Immunological investigation based on seven synthetic oligosaccharides
revealed that the α-(1→3)-linked DD-heptoglycan is a key motif for designing glycoconjugate vaccines against H. pylori, while the Ley
tetrasaccharide may rather serve as a decoy to the immune system.
10
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