Synthesis of staphylococcus aureus type 5 trisaccharide repeating unit: Solving the problem of lactamization

Article abstract of DOI:10.1021/acs.orglett.5b00031

The chemical synthesis of an orthogonally protected trisaccharide derived from the polysaccharide of Staphylococcus aureus Type 5, which is an attractive candidate for the development of immunotherapies, is described. The challenging α-fucosylation and β-mannosylation are addressed through the careful choice of protecting groups. Lactamization of a β-d-ManpNAcA moiety during deprotection was avoided by a late stage oxidation approach. Versatility of the trisaccharide was demonstrated by its transformation into a spacer-containing repeating unit suitable for immunological investigations.

Full text of DOI:10.1021/acs.orglett.5b00031

Letter
pubs.acs.org/OrgLett
Synthesis of Staphylococcus aureus Type 5 Trisaccharide Repeating
Unit: Solving the Problem of Lactamization
Ivan A. Gagarinov,†,‡ Tao Fang,†,‡ Lin Liu,† Apoorva D. Srivastava,†,‡ and Geert-Jan Boons*,†,‡
†Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States
‡Department of Chemistry, The University of Georgia, Athens, Georgia 30602, United States
S
* Supporting Information
ABSTRACT: The chemical synthesis of an orthogonally
protected trisaccharide derived from the polysaccharide of
Staphylococcus aureus Type 5, which is an attractive candidate
for the development of immunotherapies, is described. The
challenging α-fucosylation and β-mannosylation are addressed
through the careful choice of protecting groups. Lactamization
of a β-D-ManpNAcA moiety during deprotection was avoided
by a late stage oxidation approach. Versatility of the
trisaccharide was demonstrated by its transformation into a
spacer-containing repeating unit suitable for immunological investigations.
Staphylococcus aureus is a commensal of the human skin that can
cause skin and soft tissue infections (SSTIs). As many as 20% of
individuals receiving surgical and antibiotic therapy suffer from
recurrent SSTIs, which can lead to invasive disease with
bacteremia, sepsis of endocarditis.1 Of particular concern are
infections caused by antibiotic (methicillin)-resistant strains
(MRSA) for which there are limited treatment options.2
Addressing SSTIs through the development of new antibiotics
alone is not sustainable due to the continued increase in
antibiotic-resistance of MRSA isolates.3 Passive or active
immunization of patients may offer an attractive alternative for
the treatment of infections caused by MRSAs, and particularly
immunotherapies based on the capsular polysaccharide of S.
aureus are offering exciting avenues.1
There are 12 known serotypes of the S. aureus capsular
polysaccharides. However, types 5 and 8 comprise the majority of
reported isolates.4 The structures of the type 5 and 8
polysaccharides have been deduced by NMR as being →4)-β-
D-ManpNAcA-(1→4)-α-L-FucpNAc(3-OAc)-(1→3)-β-D-Fucp-
NAc-(1→ and →3)-β-D-ManpNAcA(4-OAc)-(1→3)-α-L-Fucp-
NAc-(1→3)-β-D-FucpNAc-(1→, respectively.5 Thus, the two
polysaccharides share common monosaccharides and only differ
in the location of the acetyl esters and position of an anomeric
linkage. A bivalent vaccine composed of S. aureus capsular
polysaccharide types 5 and 8 conjugated to Pseudomonas
aeruginosa exotoxin A (rEPA) gave 60% protection. However,
it failed to confer long-term protection for end stage renal disease
patients, who are often affected by these infections.6 It is thought
that well-defined oligosaccharides obtained by chemical syn-
thesis could facilitate the development of more efficacious
vaccines and therapeutic antibodies. In this respect, chemical
synthesis makes it possible to install a reactive linker for
controlled conjugation to carrier proteins, which is often
required for immunological studies. This feature is particularly
relevant for the S. aureus polysaccharides, which are highly
functionalized, and contain sensitive functional groups such as
acetyl esters that complicate conjugation chemistry. Further-
more, chemical synthesis can provide substructures and
structural analogs required for establishing structure−activity
relationships, or be used to determine minimal epitope
requirements to elicit protective immune responses.7
Previous efforts to prepare a repeating unit of S. aureus type 5
polysaccharide that can be conjugated to a carrier protein in a
controlled manner failed due to difficulties of differentiating
amino groups in the saccharide moiety and the amino propyl
linker that was needed for conjugation purposes.8 Specifically,
reduction of azido moieties of the sugar moiety led to lactam
formation of the mannosaminuronic moiety, whereas hydro-
genation of the same compound resulted in the reduction of the
azides and removal of the benzyloxycarbonyl-protecting group of
the amino propyl linker. Birch reduction had been reported for
the deprotection of a similar oligosaccharide.9 However, this
method could not be applied to the target compound due to the
presence of a base-sensitive acetyl ester.
In addition to the difficulties associated with deprotection, the
preparation of the repeating unit of an S. aureas type 5
polysaccharide (Figure 1) has a number of other synthetic
challenges. Specifically, it requires efficient syntheses of rare
monosaccharides, such as D- and L-fucosamines, and the
installation of glycosidic linkages having a 1,2-cis-configuration
including a N-acetyl-β-D-mannosaminuronic acid (β-D-Man-
pNAcA) and N-acetyl-α-L-fucosamine (α-L-FucpNAc) glyco-
sides. Furthermore, the target compound contains acetyl esters,
and therefore, base-sensitive protecting groups need to be
avoided. Finally, orthogonal protecting groups need to be
Received: January 5, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b00031
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Scheme 1. Attempted Assembly of Trisaccharide 6
an acid scavenger did prevent acceptor decomposition.13
However, the glycosylation still proceeded very slowly, and
after a reaction time of 18 h, 7 was isolated in only a trace amount
(<5%) after deprotection of C-4. An N-iodosuccinimide (NIS)/
trimethylsilyl trifluoromethanesulfonate (TMSOTf) promoted
glycosylation of 2a with 3 gave disaccharide 7 in a low yield of
32% along with unidentified byproducts possibly resulting from
acid-mediated degradation of the acceptor. The target fucoside
was obtained in an improved yield of 41% when the glycosylation
was performed under neutral conditions employing 1-benzene-
sulfinyl piperidine (BSP)/trifluoromethanesulfonic anhydride
(Tf2O) as the activator system14 at −60 °C. Use of the more
powerful diphenylsulfoxide (DPS) activator did not improve the
yield of the glycosylation. However, in each case, only α-fucoside
formation was observed, and although the yields of the
glycosylations were modest, a sufficient quantity of 7 could be
prepared to examine installation of the β-mannosamine residue.
β-Mannosides are difficult to install due to the axial C-2
substituent, which hinders the attack of an incoming nucleophile
from the β-face and the Δ-anomeric effect that stabilizes the α-
anomer.15 Recent studies indicate that preactivation of phenyl 2-
azido-2-deoxy-3-O-benzyl-4,6-benzylidene-1-thio-α-D-manno-
pyranoside 1 with the powerful DPS/Tf2O promoter system11b
offers an attractive approach for selective β-glycosylations. Thus,
activation of donor 1 was achieved at low temperature (−60 °C)
with DPS/Tf2O in the presence of 2,4,6-tri-tert-butylpyrimidine
(TTBP) in CH2Cl2. Subsequent dropwise addition of 7 followed
by slow warming to −30 °C afforded the desired β-mannoside 6
in a disappointing yield of 20%.
Figure 1. Retrosynthetic analysis.
identified that allow the preparation of a glycosyl donor and
acceptor for further oligosaccharide assembly.
We envisaged that monosaccharide building blocks 1, 2a−b,
and 3 would make it possible to assemble trisaccharide 6, which
could then be converted into the spacer containing repeating unit
of the S. aureus type 5 polysaccharide (4, Figure 1). Selective
removal of the anomeric dimethylthexylsilyl (TDS)10 ether of 6
would provide a route to a glycosyl donor whereas hydrolysis of
the benzylidene acetal followed by regioselective protection of
the C-6 hydroxyl of the resulting diol would give a potential
acceptor for oligosaccharide assembly. Furthermore, it was
anticipated that the β-D-ManpN3 glycoside of 6 could be installed
by employing glycosyl donor 1 by in situ conversion into an α-
anomeric triflate and subsequent nucleophilic displacement with
a sugar alcohol.11 Lactam formation of the β-D-ManpNAcA
moiety would be avoided by a late stage oxidation of the C-6
hydroxyl of the β-D-ManpNAc residue after reduction of the C-2
azido group, followed by acetylation of the resulting amine.
Finally, the azido moiety at C-2 of the glycosyl donors 2a−b
would allow the installation of an α-2-amino-fucoside, whereas
the 2,2,2-trichloroethoxycarbamate (Troc) moiety function at C-
2 of 3 would make it possible to form a β-2-amino-fucoside.
Monosaccharide building block 1 was synthesized by a
modification of a reported procedure11d starting from inex-
pensive N-acetyl-D-mannosamine (see Supporting Information
(SI)). Building blocks 2a−b and 3 were prepared by efficient
approaches starting from commercially available L- and D-fucose,
respectively (see SI).
It is known that the axial C-4 hydroxyl of galactosides and
fucosides are of low reactivity,16 which in the case of glycosyl
acceptor 7 is attenuated by the neighboring electron-with-
drawing acetyl ester at C-3. To improve the glycosyl accepting
properties of 7, the acetyl ester at C-3 was replaced by an
electron-donating p-methoxybenzyl (PMB) group.17 It was
Table 1. Coupling Optimization of 2b with 3
Previous studies employing 2-azido-L-fucose as a glycosyl
donor12 indicated that methyl triflate (MeOTf) is an appropriate
promoter to obtain the corresponding glycosides in good yield.
However, MeOTf-promoted coupling of 2a with 3 proceeded
sluggishly resulting in the degradation of acceptor 3, probably
due to the acid sensitivity of the anomeric TDS group (Scheme
1). The use of 2,6-di-tert-butyl-4-methylpyridine (DTBMP) as
promoter/conditions
yield
β/α
BSP, Tf2O, TTBP, CH2Cl2, −60 °C
DPS, Tf2O, TTBP, CH2Cl2, −60 °C
NIS, TMSOTf, CH2Cl2, −60 °C
NIS, AgOTf, CH2Cl2, −60 °C
NIS, TMSOTf, CH2Cl2/Et2O (4:1), −60 °C
30%
30%
73%
70%
72%
α only
α only
1/4
1/4
1/4
B
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Scheme 2. Improved Assembly of Target Trisaccharide 4
expected that the newly required glycosyl donor 2b would be
more reactive than 2a, thus providing an opportunity to improve
the challenging α-fucosylation. For the purpose of the latter
glycosylation, multiple reaction conditions were examined by
varying the solvent, temperature, and the use of additives (Table
1). It was found that a coupling of 2b with 3 in the presence of
NIS/TMSOTf in CH2Cl2 at −60 °C gave disaccharide 8 in a
much improved yield of 73% as mainly the α-anomer (α:β, 4:1).
Surprisingly, the nature of the promoter influenced the outcome
of the glycosylation and the use of BSP/Tf2O in CH2Cl2 at −60
°C gave only the α-anomer (J1,2 = 3.6 Hz), albeit in a significantly
lower yield. Next, removal of the acetyl ester of 8 using sodium
methoxide in methanol (Scheme 2) gave acceptor 9, which was
condensed with 1 using DPS/Tf2O as the promoter to give the
expected β-mannoside 10 in an excellent yield of 72%. The β-
configuration of the newly formed glycosidic linkage was
acetamido moieties, affording compound 14 in high yield.
Hydrolysis of the benzylidene acetal followed by selective
oxidation of the primary hydroxyl of the resulting diol 5 by a
modification of Huang’s one-pot TEMPO/NaOCl−NaClO2
procedure20 gave carboxylate 15 in a yield of 61% over two
steps. Finally, the remaining benzyl ethers and benzyloxy-
carbamate were removed by hydrogenation to furnish the target
trisaccharide 4.
In conclusion, a flexible orthogonally protected trisaccharide
β-D-ManpN3-(1→4)-α-L-FucpN3-(1→3)-β-D-FucpNHTroc has
been prepared in high yield with excellent anomeric selectivity
for the challenging α-fucosylation and β-mannosylation. System-
atic modification of the protecting groups of these unusual
glycosyl donors was critical for achieving high anomeric
selectivities. Late-stage regioselective oxidation of a highly
functionalized repeating unit containing β-D-ManpNAcA was
key to avoid the commonly observed lactam formation. The
strategic principles described in this letter will be relevant to the
preparation of other biologically important polysaccharides
containing β-D-ManpNAcA moieties.21 Furthermore, our
findings will guide the synthesis of larger S. aureus oligosacchar-
ide fragments required for the development of a fully synthetic
vaccine.
unambiguously confirmed by 13C NMR spectroscopy (1JCH,β
=
159.8 Hz)18 and the chemical shift of H-5 (∼3.1 ppm). The
minor α-anomer was isolated by silica gel chromatography in a
yield of 5−8% (see SI).
Encouraged by these results, the flexibility of synthon 10 was
demonstrated by performing a glycosylation−deprotection−
oxidation−deprotection sequence to prepare spacer-containing
repeating unit 4. First, the PMB ether of 10 was removed by
oxidation with 2,3-dichloro-5,6-dicyano-p-benzoquinone
(DDQ),19 which was followed by acetylation of the resulting
hydroxyl to give 6 in an 85% yield over two steps. Subsequent
cleavage of the anomeric TDS group using a hydrogen fluoride−
pyridine complex gave hemiacetal 11, which was transformed
into the corresponding N-phenyl trifluoroacetimidate 12 by
reaction with 2,2,2-trifluoro-N-phenylacetimidoyl chloride in the
presence of Cs2CO3. A TMSOTf-catalyzed glycosylation of 12
with 5-(benzyloxycarbonyl)aminopentanol in CH2Cl2/CH3CN
(1:1, v/v) gave 13 as only the β-anomer (72%). Trisaccharide 13
was then treated with Zn in a solution of THF/AcOH/Ac2O10 to
simultaneously convert the azido and Troc groups into
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental procedures and spectroscopic data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: gjboons@ccrc.uga.edu.
Notes
The authors declare no competing financial interest.
C
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ACKNOWLEDGMENTS
■
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health (GM065248, G.-J.B.).
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DOI: 10.1021/acs.orglett.5b00031
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