Total Synthesis of Tri-, Hexa- and Heptasaccharidic Substructures of the O-Polysaccharide of Providencia rustigianii O34

Article abstract of DOI:10.1002/chem.202000496

A general and efficient strategy for synthesis of tri-, hexa- and heptasaccharidic substructures of the lipopolysaccharide of Providencia rustigianii O34 is described. For the heptasaccharide seven different building blocks were employed. Special features of the structures are an α-linked galactosamine and the two embedded α-fucose units, which are either branched at positions-3 and -4 or further linked at their 2-position. Convergent strategies focused on [4+3], [3+4], and [4+2+1] couplings. Whereas the [4+3] and [3+4] coupling strategies failed the [4+2+1] strategy was successful. As monosaccharidic building blocks trichloroacetimidates and phosphates were employed. Global deprotection of the fully protected structures was achieved by Birch reaction.

Full text of DOI:10.1002/chem.202000496

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Title: Total Synthesis of Tri-, Hexa- and Heptasaccharidic Substruc-
tures of the O-Polysaccharide of Providencia rustigianii O34
Authors: Daniel B. Werz, Somayeh Ahadi, and Shahid I. Awan
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To be cited as: Chem. Eur. J. 10.1002/chem.202000496
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Total Synthesis of Tri-, Hexa- and Heptasaccharidic Substruc-
tures of the O-Polysaccharide of Providencia rustigianii O34
Somayeh Ahadi,[a]† Shahid I. Awan,[b]† and Daniel B. Werz*[a]
Abstract: A general and efficient strategy for synthesis of tri-,
hexa- and heptasaccharidic substructures of the lipopoly-
saccharide of Providencia rustigianii O34 is described. For the
heptasaccharide seven different building blocks were employed.
Special features of the structures are an α-linked galactosamine
and the two embedded α fucose units which are either branched
at positions 3 and 4 or further linked at their 2 position.
Convergent strategies focused on [4+3], [3+4], and [4+2+1]
couplings. Whereas the [4+3] and [3+4] coupling strategies failed
the [4+2+1] strategy was successful. As monosaccharidic building
blocks trichloroacetimidates and phosphates were employed.
Global deprotection of the fully protected structures was achieved
by Birch reaction.
deoxygenated, equipped with further amino groups, alkylated at
the carbon skeleton or enlarged to C8 and C9 sugars.[5]
In 2008 the carbohydrate chain of the lipopolysaccharide of
Providencia rustigianii O34 was structurally elucidated.[6] This
bacterial genus causes numerous infections in humans and is
classified into eight species (e.g. P. alcalifaciens and P.
rustigianii).[7] They are found in humans, and animal reservoirs,
but also in soil, water and sewage. In humans, Providencia
species have been isolated most commonly from urine, stool and
blood, but as well from sputum, skin, and wound cultures. The
genus Providencia includes urease-producing Gram-negative
bacteria that are responsible for a wide range of human infections.
Although most Providencia infections involve the urinary tract,
they are also associated with gastroenteritis and bacteremia.[8] In
general, an emerging issue is arising from increasing incidence of
antibiotic resistance secondary to extended-spectrum beta-
lactamase.
Introduction
The outer membrane of Gram-negative bacteria is covered with
complex lipopolysaccharides.[1] These are considered to be
virulence factors. Structurally, they consist of three distinct
domains: directly attached to the membrane is lipid A which is
connected to a core oligosaccharide (core region). The latter is
most often decorated with the so-called O-polysaccharide
(O-antigen) which differs strongly between different bacteria, but
also within different serogroups of the same bacterial species.
Thus, this coat is able to serve as a kind of fingerprint for the
detection of specific bacteria, but also for the development of
vaccine candidates.[2]
Carbohydrates serve as a perfect handle to transport specificity.
The number of possible isomers for a given number of subunits
dwarfs that of proteins by orders of magnitude.[3] Because of
several hydroxyl and/or amino groups and the possibility of two
different stereoisomers when forming the glycosidic bond a
plethora of structural isomers is possible. In addition, many
bacteria use
– besides the well-known ten mammalian
monosaccharides[4] – so-called bacterial sugars which are either
[a]
Dr. S. Ahadi, Prof. Dr. D. B. Werz
Technische Universität Braunschweig
Institute of Organic Chemistry
Hagenring 30, 38106 Braunschweig (Germany)
E-mail: d.werz@tu-braunschweig.de
Homepage: http://www.werzlab.de
Dr. S. I. Awan
[b]
Georg-August-Universität Göttingen
Institute of Organic and Biomolecular Chemistry
Tammannstraße 2, 37077 Göttingen (Germany)
These authors contributed equally to this work.
†
Supporting information for this article is given via a link at the end of
the document.
Figure 1. The structure of LPS derived from Providencia rustigianii O34.
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Because it is difficult to isolate corresponding saccharides in
acceptable purity and sufficient amount from the bacterial sources
due to their microheterogeneous character, the preparation of the
repeating subunits by chemical means is the method of choice.[9]
The schematic illustration of lipid A, a core region and the
repeating unit of the LPS derived from P. rustigianii O34, are
depicted in Figure 1. Although no special bacterial
monosaccharides are found in the glycan it shows some structural
challenges. In contrast to mammalian oligosaccharides[4] in which
fucose is only known as a terminal unit, we encounter two
α-fucoses embedded in the chain. One of them is connected to
the next monosaccharidic unit via its 2-hydroxy group while the
other is branched with further monosaccharide residues at the 3-
and 4-hydroxy groups. As terminal sugars at the non-reducing
end, β-D-glucuronic acid and α-D-galactosamine are found. In this
paper, we describe our synthetic endeavors to access tri-, hexa-
and heptasaccharidic substructures of the O-polysaccharide
found on the outer surface membrane of Providencia rustigianii
O34. Such fragments conjugated to a protein should be highly
useful tools to elicit carbohydrate-specific antibodies against
these bacteria.
Results and Discussion
The target structure 1 was equipped with a pentenyl chain at the
reducing sugar to have a handle for further modification, or as an
attachment point for proteins (Scheme 1). Many retrosynthetic
strategies can be envisioned for the desired compound. We
started our attempts with [4+3]- and [3+4]-couplings, but failed to
achieve the glycosidic bonds between Fuc and Man or Man and
Fuc, respectively (for more details see Supporting Information).
Finally, a [4+2+1] coupling strategy was more successful. The
retrosynthetic analysis of 1 is depicted in Scheme 1. In total,
seven different building blocks 4-10 are necessary. As temporary
protecting groups Fmoc, PMB and Ac are used whereas Bn and
Piv groups permanently protect the hydroxyl groups. The amine
functionalities are silenced by either trichloroacetamide (TCA)
protection or aremasked as azides.
Scheme 1. Different retrosynthetic strategies (failed [3+4] and [4+3]) and our
successful [4+2+1] strategy to build up the heptasaccharide unit.
Our synthetic endeavor started with the preparation of the
literature-known n-pentenyl D-glucosamine acceptor 5.[10] The
second building block, glucosyl dibutyl phosphate 6, was
prepared in a one-pot procedure from perbenzylated D-glucal 11
(Scheme 2). Dimethyldioxirane (DMDO)[11] is able to attack the
electron-rich enol ether in a facially selective manner to form a
highly reactive acetal epoxide which is easily opened under acidic
conditions by dibutylphosphate. The emerging hydroxy group was
converted by FmocCl into the respective carbonate. The carbonyl
as neighboring-participating group ensures the β-selectivity in the
glycosylation reaction.
Scheme 2. Synthesis of the D-glucosyl phosphate donor 6.
The well-known D-mannosyl phosphate 8 was prepared in a few
steps according to literature methods from commercially available
mannose (see Supporting Information).[12]
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The two fucose building blocks were prepared from a common
literature-known 3,4-isopropylidene-protected fucose 13.[13] Both
fucose moieties in the target structure are α-linked; thus, it is
prerequisite that no neighboring-participating group is located in
position 2. Since one of the fucose units is linked at the 2-OH
group a removable ether group as temporary protecting group is
crucial. Therefore, we relied on p-methoxybenzyl ether (PMB)[14]
which was installed in 73% yield. Cleavage of the acetonide in 14
was achieved by using an indium-based procedure.[15] Besides
the non-participating group in position 2, an installation of
electron-withdrawing groups in position 3 and 4 is another
possibility to further increase α-selectivity in the glycosylation
reaction.[16] Thus, pivaloate esters were installed. The explanation
for these groups being beneficial are various; there are reports
that suggest these groups are remote participating groups[17]
whereas others only emphasize the electron-withdrawing effect.
Nevertheless, their utility to achieve high α-selectivity has been
demonstrated in many cases. The cleavage of the anomeric
pentenyl protecting group was achieved by NBS in aqueous
conditions. The resulting hemiacetal was converted into the
Schmidt donor 17 in 68% yield over two steps. Later, we found
that the respective phosphate showed a better behavior in the
glycoslation reaction; thus, the former donor was further
transformed into the fucosyl phosphate 7 (Scheme 3).
The other fucose unit is branched with two different residues
attached to position 3 and 4. In our [4+2+1] strategy this fucose
serves as a starting point (i.e. as glycosyl acceptor) for a
disaccharide synthesis; therefore, only one temporary protecting
group is required. 2-Benzyl protection ensures the α-selectivity.
Acidic conditions cleave the isopropylidene of 13. A regioselective
protection of the 4-hydroxy group was achieved by the reaction
with methyl orthoformate in the presence of p-TsOH leading to an
intermediate formation of the corresponding orthoester. The latter
was opened by AcOH (80%) [18, 19] in such a way that only the axial
acetate results; consequently, the desired fucosyl acceptor 9 was
obtained in quantitative yield over two steps (Scheme 3).
Scheme 3. Synthesis of the L-fucosyl phosphate donor 7 and L-fucose acceptor
9.
The terminal galactosamine building block was obtained via an
azidonitration procedure[20] starting from perbenzylated galactal
18. In three steps the respective trichloroacetimidate was
obtained. In some instances, glycosyl phosphates gave better
results in our hands than the corresponding trichloroacetimidates;
thus, we converted 19 into its phosphate analog 20 in 87% yield
(Scheme 4).
Scheme 4. Synthesis of the D-galactosyl trichloroacetimide 19 and phosphate
20.
The final building block 27 is accessible from literature-known triol
22.[21] Treatment of the triol with benzaldehyde dimethyl acetal
under the catalytic influence of p-TsOH provided benzylidene
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acetal 23 (Scheme 5). Protection of the 2-hydroxy group by
pivaloate, followed by subsequent regioselective ring-opening of
the benzylidene acetal using a borane-THF complex in the
presence of Bu2BOTf furnished alcohol 25. Transformation to the
corresponding glucuronic acid was achieved under oxidizing
conditions, the emerging acid functionality was immediately
protected by methylation with methyl iodide under basic
conditions. Finally, the anomeric protecting group was removed
by the action of cerium ammonium nitrate (CAN); the hemiacetal
was transformed into trichloroacetimidate 4. Only one anomer
was formed; the small 3J1,2 coupling constant of 3.6 Hz suggests
the generation of the α anomer. The respective phosphate 27 was
obtained in 75% yield by the reaction of 4 with dibutyl phosphate.
3 in 80% yield. A subsequent saponification removed the acetate
in position 2 of the mannose unit affording 32 (Scheme 6).
Scheme 6. Synthesis of the tetrasaccharide acceptor 32.
Initially, we tried a [4+3] approach to build up the heptasaccharide.
Thus, we prepared a branched trisaccharide building block which
contains fucose, galactosamine and glucuronic acid. We started
to investigate the formation of this α-linked disaccharide by using
a more easily available perbenzylated galactosamine donor 20
with an azide moiety in position 2. Although ether was used as
solvent which should favor the desired α-selectivity, it was poor
(α/β = 1.3/1) (Scheme 7). Thus, a modified less reactive building
block was prepared starting from the respective 3,6-dibenzylated
galactal. Protection of the 4-hydroxy group by pivaloate and
azidonitration, followed by reductive nitrate cleavage led to
hemiacetal 37 which was further transformed into the
trichloroacetimidate 10 (Scheme 8).
Scheme 5. Synthesis of the D-glucosyl trichloroacetimide 4 and phosphate
donor 27.
With these building blocks in hand we started to assemble the
heptasaccharide. Galactosyl acceptor 5 and glucosyl phosphate
6 were coupled under Lewis-acidic conditions applying TMSOTf
as promotor leading to the (1→3)-linked disaccharide 28 in 80%
yield (Scheme 6). High β-selectivity was observed due to
anchimeric assistance of the Fmoc group. Deprotection of the
latter using piperidine generated disaccharide 29. The
glycosylation of 29 with fucosyl phosphate 7 in the presence of
TMSOTf led to trisaccharide 30 with complete α-selectivity.
Because of the acidic conditions the PMB group was cleaved to
some extent,[22] nevertheless the PMB-protected trisaccharide
was obtained as major product. This compound was treated with
CAN in aqueous medium to afford trisaccharide acceptor 31.
Glycosylation with mannosyl phosphate 8 yielded tetrasaccharide
Scheme 7. Synthesis of the disaccharide 33.
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For the [4+3] coupling a plethora of different conditions were
tested (see Supporting Information). All attempts remained
unsuccessful and tetrasaccharide acceptor was recovered either
unaffected or with partial decomposition. The trisaccharide donor
often decomposed to its hydrolyzed form or was isolated as the
trehalose-like 1,1-linked dimerization product. Steric hindrance of
the branched trisaccharide seems to be too high for a successful
glycosylation. As an alternative we also performed a [3+4]
coupling starting with
a
trisaccharide acceptor and
a
tetrasaccharide donor; However, these attempts were also
unsuccessful (see Supporting Information).
After these failures, we redesigned our strategy towards a [4+2+1]
coupling. The monosaccharidic building blocks are the same as
previously described (see Scheme 1). Instead of a trisaccharide
donor, we prepared disaccharide trichloroacetimidate 2 from
pentenyl disaccharide 38 described above (Scheme 10). This
trichloroacetimidate was united with the tetrasaccharide (as
described in Scheme 6). In contrast to the previous attempts to
establish that linkage this [4+2] coupling afforded the desired
hexasaccharide 47 in 67% yield and based on recovered starting
materials even with up to 90% yield (Scheme 11). The
subsequent transesterification under basic conditions removed
the acetate in position 4 of the second fucose moiety to afford 48.
Scheme 8. Synthesis of the D-galactosyl donor 10.
With the more electron-poor galactosamine donor in hand we
performed again the glycosylation to the disaccharide. Now,
complete α-selectivity was obtained. The following reactions were
carried out with both the benzylated disaccharide (as described in
Scheme 7) and the disaccharide with a Piv group in the 4-position.
The disaccharides 33α and 38 were hydrolyzed under basic
conditions (in the case of pivaloate strong basic conditions should
be avoided); the emerging hydroxy groups were subsequently
reacted with methyl glucoronate 4 under Lewis acidic conditions
to furnish trisaccharides 41 and 42, respectively. Cleavage of the
pentenyl group generated the hemiacetals which were
transformed via the trichloroacetimidates into the corresponding
phosphates 45 and 46 (Scheme 9).
Scheme 10. Synthesis of disaccharide trichloroacetimidate 2.
To build up the heptasaccharide, extensive screening with
respect to the promotor was necessary. The glycosidic bonds to
the fucose residues tend to be cleaved under strong Lewis acidic
conditions. Thus, it was very important to choose carefully the
Lewis acid for the final glycosidic bond formation. With BF3•OEt2
only slow conversion was observed and a large amount of
unreacted hexasaccharide acceptor was recovered from the
reaction mixture. When TMSOTf was used to catalyze the
glycosylation reaction increased reactivity was observed, but it
was accompanied by decomposition of the starting materials. In
contrast to TMSOTf, a milder Lewis acidic promotor such as
TESOTf produced the desired product in a moderate yield.
Further optimization of other parameters showed that this very
sensitive glycosylation step gives the best yield after stirring for
24 h at 0 °C (Scheme 11). It is noteworthy that slow addition of
the diluted activator was crucial (see Supporting Information).
Scheme 9. Synthesis of the trisaccharide phosphates 45 and 46.
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Scheme 11. Synthesis of heptasaccharide 49 using a [4+2+1] strategy.
Finally, we used size-exclusion gel-permeation HPLC to obtain
the pure product 49 (see Supporting Information). As side
products, smaller parts of the heptasaccharide were found in all
cases. The glycosidic bonds to fucose units are not as stable as
to Glc, Gal or Man. Under the influence of strong Lewis or
Brønsted acids they tend to be cleaved because the emerging
positive charge is much better stabilized in these 6-deoxy sugars
than in the 6-oxygenated counterparts.
With the tri-, hexa- and heptasaccharides 31, 47, and 49 in hand
we performed studies with respect to their global deprotection.
Hydrogenation of the larger structures was associated with
incomplete removal of the Bn groups; therefore, we employed the
Birch reaction to achieve our goal. It is well-known that the TCA
group often causes problems in Birch reactions because the
relatively acidic N-H bond gets deprotonated and the emerging
anion acts as a good nucleophile.[23] As a result, the adjacent
glycosidic bond might break. Thus, we first used zinc-copper
couple as a reducing agent to convert the TCA group into its
acetamide analog, followed by acetylation of other emerging
amino groups since azide moieties are also reduced using this
procedure. Global deprotection was obtained under Birch
conditions. For simpler purification the naked sugars were
acetylated and then treated with sodium methoxide in methanol.
The desired target compounds 51 and 53, respectively, were
obtained in pure form after dialysis and lyophilization (Scheme 12).
Scheme 12. Global deprotection to tri- and hexasaccharides 51 and 53.
Encouraged by these results, we applied the optimized conditions
to access the deprotected heptasaccharide. Firstly, acetamides
were generated from the TCA-protected amine and the azide,
respectively. To avoid epimerization at C-5 of the glucuronic acid
under Birch conditions, we transformed the ester to the
corresponding carboxylic acid. Global deprotection by Birch
reaction, subsequent acetylation and deacetylation yielded the
final product 1 which was confirmed by high-resolution mass
spectrometry and 1H NMR spectroscopy including HSQC
(Scheme 13). Because of the very small amount we were unable
to record a proper 13C NMR spectrum. Although we performed a
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Scheme 13. Synthesis of heptasaccharide 1.
Conclusions
Herein, we presented the first syntheses of the tri-, hexa- and
heptasaccharidic substructures of the O-polysaccharide in
Providencia rustigianii O34. The polymeric structure consists of a
repeating unit with seven different monosaccharide moieties; thus,
seven different monosaccharidic building blocks were used.
Because relatively labile α-linked fucosyl residues are embedded
in the structure special care is needed for the choice of the
glycosylation conditions. Lewis acids which are too harsh led to a
decomposition of the oligomer by cleaving α-fucosidic bonds. The
reducing end of all structures was equipped with a pentenyl
handle, which can be used for attachment to surfaces and
proteins. In future, these structures will be employed to elicit
carbohydrate-specific antibodies that might be useful for the
detection of Providencia rustigianii O34 or even for vaccination
studies.
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Acknowledgements
[9]
S. A is grateful to the Alexander von Humboldt Foundation (Georg
Forster Research Fellowship) for financial support. S. I. A. thanks
the Higher Education Commission of Pakistan for a Ph.D.
Fellowship. We thank Gwyndaf Oliver (TU Braunschweig) for
proofreading the manuscript.
Keywords: Providencia rustigianii • lipopolysaccharide (LPS) •
oligosaccharides • glycosylation • fucose
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Embedded fucoses: First syntheses
of substructures of the O-polysaccha-
ride of Providencia rustiginii O34 are
presented. The repeating unit is a
heptasaccharide with seven different
monosacchride units.
Somayeh Ahadi, Shahid I. Awan, and
Daniel B. Werz*
Page No. – Page No.
Total Synthesis of Tri-, Hexa- and
Heptasaccharidic Substructures of
the O-Polysaccharide of Providencia
rustigianii O34
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