Progress toward developing a carbohydrate-conjugate vaccine against Clostridium difficile ribotype 027: Synthesis of the cell-surface polysaccharide PS-I repeating unit

Article abstract of DOI:10.1039/c1cc13614c

Clostridium difficile strain ribotype 027 is a hypervirulent pathogen that is responsible for recent, severe outbreaks of serious nosocomial infections. As a foundation for the development of a preventative carbohydrate-based vaccine, we have synthesized a pentasaccharide cell wall repeating unit from PS-I unique to this strain, by the linear assembly of four monosaccharide building blocks.

Full text of DOI:10.1039/c1cc13614c

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Progress toward developing a carbohydrate-conjugate vaccine against
Clostridium difficile ribotype 027: synthesis of the cell-surface
polysaccharide PS-I repeating unitwz
Christopher E. Martin,ab Markus W. Weishauptab and Peter H. Seeberger*ab
Received 17th June 2011, Accepted 21st July 2011
DOI: 10.1039/c1cc13614c
Clostridium difficile strain ribotype 027 is a hypervirulent
pathogen that is responsible for recent, severe outbreaks of
serious nosocomial infections. As a foundation for the develop-
ment of a preventative carbohydrate-based vaccine, we have
synthesized a pentasaccharide cell wall repeating unit from
PS-I unique to this strain, by the linear assembly of four
monosaccharide building blocks.
pentasaccharide phosphate repeating unit with the structure
[- 4)-a-Rhap-(1 - 3)-b-Glcp-(1 - 4)-[a-Rhap-(1 - 3]-a-
Glcp-(1 - 2)-a-Glcp-(1 - P] and, so far, has been found to be
expressed by C. difficile ribotype 027 only.6 The branched
pentasaccharide repeating unit 1 of PS-I is shown in Fig. 1.
Units A and B are 1 - 2 linked a-Glc residues, wherein A is
equipped with the aminopentyl linker at the reducing terminus,
and B constitutes the branching point. B connects to a-Rha
residue D at C-3 and b-Glc residue C at C-4. Finally, residue
D0 is the terminal a-Rha, linked to C-3 of C.
Here we report the first total synthesis of pentasaccharide 1,
the synthetic challenge was met by devising a linear strategy
using a set of building blocks 2–7 (Fig. 1). This linear strategy
allowed for the installation of the challenging 1,2-cis glycosidic
linkages of both residues A and B early in the synthetic
pathway by using building blocks 2 and 3. Formation of the
linkage between C and the AB fragment was investigated using
three different glycosylating agents of C, namely thioglycoside
Clostridium difficile is a Gram-positive, spore-forming anae-
robic bacterium that colonizes the intestinal tract of humans
thus leading to C. difficile infections (CDI).1 CDI have become
the most commonly diagnosed cause of hospital-acquired
diarrhea, particularly in risk groups including the elderly
and immunodeficient patients, as well as those receiving
antibiotic treatment. A steep rise in the incidence of CDI over
the past decade is attributed to the emergence of the hypervirulent,
and now predominant strain ribotype 027 that causes epidemic
outbreaks with increased morbidity, mortality and high rates
of relapse.2 These infections significantly increase the treatment
costs of patients, particularly in the case of recurring CDI.3
Prevention of CDI is therefore desirable, and vaccination of
risk groups may be a useful and cost-efficient means to avoid
future infections. Although models have shown that vaccination
against C. difficile should be economically feasible,4 a vaccine
has not yet been developed.
Carbohydrates exposed on the cell-surface of pathogens are
often immunogenic and constitute potential candidates for
vaccine development. When covalently connected to a carrier
protein, a carbohydrate antigen can elicit long lasting, T-cell-
dependent protection.5 The chemical structures of two
C. difficile cell-surface polysaccharides, PS-I and PS-II were
recently elucidated6 Initially, the focus was directed towards
the PS-II hexasaccharide repeating unit antigen7 because it is
common to several C. difficile strains. PS-I consists of a
a Max Planck Institute of Colloids and Interfaces, Department of
Biomolecular Systems, Am Muhlenberg 1, 14476 Potsdam, Germany.
¨
E-mail: peter.seeberger@mpikg.mpg.de
b Freie Universitat Berlin, Institute for Chemistry and Biochemistry,
¨
Arnimallee 22, 14195 Berlin, Germany
w This article is part of the ChemComm ‘Glycochemistry and
glycobiology’ web themed issue.
z Electronic supplementary information (ESI) available: Experimental
details and full spectroscopic data for all new compounds. See DOI:
10.1039/c1cc13614c
Fig. 1 Retrosynthesis of the PS-I pentasaccharide repeating unit 1.
c
10260 Chem. Commun., 2011, 47, 10260–10262
This journal is The Royal Society of Chemistry 2011

in four steps from glucose pentaacetate 10.10 We found that use
of the nontoxic and odorless 2-methyl-5-tert-butylthiophenol
group11 ensures exclusive b-anomer formation of thioglucosides
and results in storage-stable monomer units. The adaptable
thiol group is readily converted to phosphates or imidates,
thus allowing creation of the series of glycosylating agents 3–6.
The protecting group pattern of 13 enables the orthogonal
protection of all hydroxyl groups of the hexose ring. This
feature was exploited in the synthesis of building block 3,
where a non-participating benzyl group was installed at C-2 of
intermediate 14 to favor the formation of the a-glycosidic
linkage between the A and B fragments. Subsequent placement
of the 3-O-Fmoc-protection furnished compound 15. Finally,
regioselective opening of the 4,6-O-benzylidene acetal with
TES–Tf OH and protection of the free 4-hydroxyl gave
orthogonally-protected building block 3.
Scheme 1 Synthesis of building block 2. Reagents and conditions: (a)
NaH, NAPBr, DMF, 0 1C to rt, 92%; (b) HO(CH2)5NBnCbz, NIS,
TfOH, toluene/dioxane, ꢀ40 1C to ꢀ20 1C; (c) DDQ, DCM, H2O,
35% over 2 steps.
4, N-phenyl trifluoroacetimidate 5 and glycosyl phosphate 6.
In order to minimize the number of deprotection and glycosylation
steps, both Rha residues D and D0 were added to the intermediate
trisaccharide ABC in a single bis-glycosylation reaction with
building block 7.
The synthesis of building block 2 is depicted in Scheme 1.
The latent amine at the reducing end was introduced by union
of thioglucoside 9 and the linker.8 Thereby, 2-naphthylmethyl
(NAP) served as a non-participating temporary protecting
group at C-2 in order to achieve formation of the a-glycosidic
linkage. Subsequent 3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) mediated cleavage of the NAP ether resulted in build-
ing block 2.9
Preparation of differentially-protected thioglycoside 4 from
13 followed a similar route. In anticipation of the formation of
a 1,2-trans linkage between the B and C saccharide fragments,
a participating benzoyl group was installed at the C-2 hydroxyl
of intermediate 17. During TBAF-mediated desilylation of 17,
careful control of the TBAF:AcOH ratio was essential to
prevent benzoyl-migration from C-2 to C-3. Fmoc-protected
thioglycoside 4 was further diversified to glycosyl imidate
5 and glycosyl phosphate 6. Building blocks 4–6 were individu-
ally evaluated for their reactivity towards the AB disaccharide
fragment in assembly of the ABC trisaccharide. The terminal
D and D0 fragments of the pentasaccharide were provided by
building block 7. Synthesis of this rhamnosyl unit commenced
with the bis-benzoylation of p-methoxyphenyl glycoside
19 (Scheme 3).12
All four building blocks 3–6 originate from the key inter-
mediate 13 (Scheme 2). This common precursor is synthesized
CAN-mediated removal of the anomeric p-methoxyphenyl
group yielded the free lactol that was immediately converted
into rhamnosyl N-phenyl trifluoroacetimidate 7.13
Assembly of the pentasaccharide target was achieved in
seven linear steps by combining the monosaccharide building
blocks in sequence (Scheme 4). The 1,2-cis glycosidic linkage
between residues A and B was formed using nucleophile 2 and
glycosylating agent 3. Disaccharide 21 was obtained in good
yield and stereoselectivity when NIS and Tf OH in Et2O was
employed as promoter system. Selective cleavage of the
levulinic ester with hydrazine hydrate in pyridine/AcOH, did
not compromise the integrity of the Fmoc-group but cleanly
produced disaccharide acceptor 22. Installation of the next
glycosidic linkage between C and the AB fragment to form
trisaccharide 23 was attempted using the different building
blocks 4–6 and the three reactions were screened for their
Scheme 2 Synthesis of monosaccharide building blocks 3–6. Reagents
and conditions: (a) 2-methyl-5-tert-butylthiophenol, BF3ꢁOEt2, DCM,
85%; (b) NaOMe, MeOH, rt; (c) benzaldehyde dimethyl acetal, CSA,
MeCN, 87% over 2 steps; (d) TBS-Cl, imidazole, DMF, 0 1C, 69%; (e)
NaH, BnBr, DMF, 0 1C to rt; (f) 1 M TBAF in THF, 0 1C to rt, 93%
over 2 steps; (g) Fmoc-Cl, pyridine, DCM, 95%; (h) TES, Tf OH,
DCM, 4 A MS, ꢀ78 1C, 73%; (i) Lev2O, pyridine, DCM, 3 days, 79%;
(j) BzCl, DMAP, pyridine, 70 1C, 88%; (k) TBAFꢁ3H2O, AcOH, DMF,
35 1C, 91%; (l) Fmoc-Cl, pyridine, DCM, 96%; (m) NIS, AgOTf,
TTBP, MeCN, H2O; (n) CF3C(NPh)Cl, Cs2CO3, DCM, 67% over 2
steps; (o) HOPO(OBu)2, NIS/Tf OH, DCM, 4 A MS, 0 1C, 81%.
Scheme 3 Synthesis of rhamnosyl building block 7. Reagents and
conditions: (a) BzCl, DMAP, pyridine, DCM, 0 1C to rt, 97%; (b)
CAN, MeCN, H2O; (c) CF3C(NPh)Cl, Cs2CO3, DCM, 74% over 2
steps.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10260–10262 10261

Scheme 4 Synthesis of 1. Reagents and conditions: (a) 3, NIS/TfOH, Et2O, ꢀ35 1C to ꢀ10 1C, 70%; (b) N2H4ꢁH2O, AcOH/pyridine, DCM, 94%;
(c) 6, TMSOTf, DCM, 4 A MS, ꢀ35 1C to ꢀ7 1C; (d) NEt3, DCM, rt, 38% over 2 steps; (e) 7, TMSOTf, DCM, 4 A MS, ꢀ30 1C to ꢀ15 1C, 81%;
(f) NaOMe, THF/MeOH, 50 1C; (g) H2, 10% Pd/C, MeOH, H2O, AcOH, 61% over 2 steps.
efficiency. Use of thioglycoside 4 and N-phenyl trifluoroacetimidate
Notes and references
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was achieved only following Fmoc cleavage to yield 24. Finally,
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7 in the presence of TMSOTf to add both rhamnose residues.
Final deprotection of 25 required two transformations:
saponification of the benzoate esters and catalytic hydrogenation
of the aromatic groups gave pentasaccharide 1.
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1 and native PS-I6 showed overall good agreement, chemical
shifts of signals corresponding to residues B, C and D were
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not present in the synthetic structure (see ESI for more
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10262 Chem. Commun., 2011, 47, 10260–10262
This journal is The Royal Society of Chemistry 2011