First synthesis of C. difficile PS-II cell wall polysaccharide repeating unit

Article abstract of DOI:10.1021/ol1026188

Clostridium difficile is the most commonly diagnosed cause of nosocomial diarrhea with increasing incidence and mortality among elderly and hospitalized patients. We report the first synthesis of the surface polysaccharide PS-II repeating unit and its non

Full text of DOI:10.1021/ol1026188

ORGANIC
LETTERS
2011
Vol. 13, No. 3
378-381
First Synthesis of C. difficile PS-II Cell
Wall Polysaccharide Repeating Unit
Elisa Danieli,† Luigi Lay,‡ Daniela Proietti,† Francesco Berti,† Paolo Costantino,†
and Roberto Adamo*,†
NoVartis Vaccines & Diagnostics, Vaccine Chemistry Department, Via Fiorentina 1,
53100 Siena, Italy, and UniVersity of Milan, Department of Organic and Industrial
Chemistry, CISI and ISTM-CNR, Via G. Venezian 21, 20133 Milan, Italy
roberto.adamo@noVartis.com
Received October 28, 2010
ABSTRACT
Clostridium difficile is the most commonly diagnosed cause of nosocomial diarrhea with increasing incidence and mortality among elderly
and hospitalized patients. We report the first synthesis of the surface polysaccharide PS-II repeating unit and its nonphosphorylated analogue,
with a linker for conjugation, via a (4 + 2) convergent approach from a common AB(D)C tetrasaccharide intermediate.
Clostridium difficile is a Gram-positive spore-forming anaer-
obic bacterium, which is considered the most important
definable cause of nosocomial diarrhea.1,2 Since its descrip-
tion in 1978 as a cause of antimicrobial-associated diarrhea,
colitis, and pseudomembranous colitis (PMC), the interest
in this pathogen is growing due to its impact on morbidity
and mortality in the elderly and among hospitalized patients.3
The incidence of C. difficile infection (CDI) is rapidly
increasing in the US and Canada,4 where a recent study reported
an attack rate of 22.5 cases per 1000 hospital admissions, which
was associated with a significantly high mortality rate of 6.9%.3,5
The most virulent strain is considered the ribotype 027 or
North American pulsotype 1 (NAP1), which caused out-
breaks in 16 European countries in 2008.2 Current treatments
for CDI are suboptimal, with up to 20% of treated patients
failing to respond to antibiotics and relapses occurring in
up to 25% of cases after initial clinical resolution.6 Health-
related costs for afflicted people are significant and estimated
as $4000/case in the US.7 Treatment failures and recurrences
with antibiotics are emphasizing the need for the discovery
of new preventative agents using vaccination based on either
protein or carbohydrate antigens.8
Monteiro’s group recently analyzed the cell wall polysac-
charide of C. difficile rybotype 027 and two additional strains,
MOH900 (classified as NAP2) and MOH718.9 Two different
structures were identified, named PS-I and PS-II. However,
PS-II is the only structure occurring in all strains. This finding
strongly suggests that PS-II is a conserved surface antigen,
† Novartis Vaccines & Diagnostics.
‡ University of Milan.
(5) Loo, V. G.; Poirier, L.; Miller, M. A.; Oughton, M.; Libman, M. D.;
Michaud, S.; Bourgault, A.-M.; Nguyen, T.; Frenette, C.; Kelly, M.; Vibien,
A.; Brassard, P.; Fenn, S.; Dewar, K.; Hudson, T. J.; Horn, R.; Rene´, P.;
Monczak, Y.; Dascal, A. N. Engl. J. Med. 2005, 353, 2442.
(6) Redelings, M. D.; Sorvillo, F.; Mascola, L. Emerg. Infect. Dis. 2007,
13, 1417.
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2010, 825
.
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6, 251
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Goorhuis, B.; Kuijper, E. J.; Wilcox, M. H. Clin. Microbiol. ReV. 2010,
23, 529.
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Dis. 2002, 34, 346.
(4) Pe´pin, J.; Valiquette, L.; Alary, M.-E.; Villemure, P.; Pelletier, A.;
Forget, K.; Pe´pin, K.; Chouinard, D. Can. Med. Assoc. J. 2004, 171, 466.
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10.1021/ol1026188  2011 American Chemical Society
Published on Web 12/29/2010

which may be advantageous in the development of a
carbohydrate-based anti C. difficile vaccine. PS-II is com-
posed of a hexasaccharide phosphate repeating unit: [f6)-
ꢀ-D-Glcp-(1f3)-ꢀ-D-GalpNAc-(1f4)-R-D-Glcp-(1f4)-[ꢀ-
D-Glcp-(1f3]-ꢀ-D-GalpNAc-(1f3)-R-D-Manp-(1fP].
To the best of our knowledge, the immunogenicity of such
polysaccharide and consequently the possibility of preparing
a glycoconjugate vaccine have not hitherto been investigated.
The synthetic approach would be considerably appropriate
to obtain well-defined structures to be tested as anti C.
difficile vaccines and to circumvent fermentation and isola-
tion of the polysaccharide from spore-forming bacteria.10
Here we report the first synthesis of the hexasaccharide
PS-II repeating unit 2 and its nonphosphorylated analogue
1 (Figure 1). Both oligosaccharides were synthesized with
both for the synthesis of tetrasaccharide 5 and the construc-
tion of hexasaccharide 1. In addition, the challenging
insertion of the 1,2-cis glycosidic linkage between residues
D and B could be carried out in an early stage of our work.
On the other hand, the preparation of the phosphorylated
hexasaccharide 2 required disaccharide donor 4, which differs
from 3 by a further selectively removable group at the
primary hydroxyl of the C′ unit.
We employed the N-trichloroethoxycarbonyl (Troc) par-
ticipating group for the amino group protection in the
galactosamine units of 3 and 412 to ensure the formation of
1,2-trans glycosidic linkages.
The synthesis of 2,4,6-tri-O-benzylated mannoside 9,
bearing the R-oriented anomeric linker, was carried out as
illustrated in Scheme 1.
Scheme 1. Synthesis of the Mannoside Building Block 9
After glycosylation of commercially available benzyl N-(3-
hydroxypropyl)carbamate with donor 10,13 the resulting
mannoside 1114 was deacetylated and converted into the 2,3-
O-isopropylidene derivative 12 by 2,3:4,6-bis-isopropylide-
nation followed by selective monohydrolysis.15 Benzylation
of diol 12, using NaOH as a base under phase-transfer
conditions in order to prevent concomitant N-benzylation of
the linker,13 and subsequent isopropylidene acetal hydrolysis
Figure 1. Retrosynthetic analysis of PS-II hexasaccharide.
an O-linked aminopropyl spacer at the reducing end suitable
for conjugation to a carrier protein, which is a fundamental
step to make poorly immunogenic carbohydrates able to
induce a T cell dependent response.11 According to our
retrosynthetic analysis, target hexasaccharides 1 and 2 can
be assembled by a (4 + 2) convergent approach from a
common tetrasaccharide intermediate AB(D)C 5 (Figure 1).
This strategy features disaccharide 3 as a key intermediate
(10) For reviews on oligosaccharide-based glycoconjugate vaccines, see:
(a) Astronomo, R. D.; Burton, D. R. Nat. ReV. Drug. DiscoV. 2010, 9, 308.
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D. B. Nature 2007, 446, 1046. (d) Vliegenthart, J. F. G. FEBS Lett. 2003,
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S.; Fabbri, B.; Giannini, S.; Giannozzi, A.; Magagnoli, C.; Proietti, D.;
Norelli, F.; Rappuoli, R.; Ricci, S.; Costantino, P. Vaccine 2009, 26, 2284.
(12) Z. Zhang, G. M. Carbohydr. Res. 1996, 280, 251. Ellervik, U.;
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Org. Lett., Vol. 13, No. 3, 2011
379

afforded compound 14. Regioselective p-methoxybenzylation
at 3-OH of 14 using the stannylene acetal protocol allowed
the benzylation of the axial 2-hydroxyl.16 Finally, standard
oxidation of 16 with 3-dichloro-5,6-dicyano-1,4-benzo-
quinone provided the 3-OH mannosyl acceptor 9.
as a Lewis acid required dichloromethane-hexane as a
solvent mixture to circumvent formation of the 1-N-
trichloroacetamide,19 which was the predominant byprod-
uct when the reaction was performed in only dichlo-
romethane. Disaccharide 3 could be obtained in a
satisfactory 82% yield, whereas the preparation of the
closely related compound 19 from donor 1820 proceeded
in lower yield (52%) since the formation of substantial
amount of the 1-N-trichloroacetamide byproduct could not
be avoided. Disaccharide 19 was then regioselectively
6-O-deacetylated by mild transesterification with NaOMe
at pH 9 and 0 °C, allowing the straightforward introduction
of the tert-butyldiphenylsilyl protecting group to afford
compound 4.
The syntheses of disaccharide donors 3 and 4 were achieved
starting from the N-Troc galactosamine acceptor 7 (Scheme 2),
Scheme 2. Syntheses of the Disaccharide Donors 3 and 4
Thioglycoside 3 was used as a donor for glycosylation of
the acceptor 9 promoted by NIS-TfOH, giving trisaccharide
21 in 77% yield (Scheme 3).
Compound 21 was subjected to regioselective opening of
the benzylidene acetal by borane-trimethylamine complex
and BF3·Et2O21 to directly furnish the trisaccharide acceptor
22 (80% yield). Gratifyingly, the glycosylation of 22 with
ethylthioglycoside 822 in toluene-dioxane using NIS-
TfOH as promoters permitted the stereoselective introduction
of the R-linkage and provided tetrasaccharide 23 in 89%
yield. The R configuration of the newly formed glycosidic
1
bond was confirmed in H NMR spectrum (CDCl3, 400
MHz) by a doublet appearing at 5.11 ppm corresponding to
H-1D with J1,2 ) 2.3 Hz (see the Supporting Information).
A second regioselective ring-opening step provided ef-
ficiently tetrasaccharide acceptor 5 in 95% yield. Hexasac-
charide 24, leading to the nonphosphorylated analogue of
PS-II repeating unit, was then completed by glycosylation
of the tetrasaccharide 5 with the disaccharide donor 3 in
moderate 50% yield (Scheme 3). Conversion of the Troc
group into acetamide was carried out by basic hydrolysis,
which led to concomitant removal of the O-acetyl esters,
followed by N-acetylation.
obtained from the known compound 1717 by deacetylation and
introduction of the 4,6-O-benzylidene acetal.
Glycosylation of monosaccharide acceptor 7 with
trichloroacedimidate donor 618 in the presence of TMSOTf
Scheme 3. Assembling of the Hexasaccharide 1
380
Org. Lett., Vol. 13, No. 3, 2011

Scheme 4. Assembly of the Hexasaccharide 2
Hydrogenation of hexasaccharide intermediate 25 by flow
chemistry, utilizing a 10% Pd-C cartridge, gave the first
target molecule 1.
Since charged groups are often important epitopes for
bacterial polysaccharides, the role of the phosphate group
occurring in the PS-II repeating unit is an important issue to
be addressed.
Accordingly, the synthesis of phosphorylated hexasaccharide
2 was approached by glycosylation of tetrasaccharide acceptor
5 with disaccharide donor 4 in 62% yield (Scheme 4).
After Troc group removal with 0.3 M NaOH from
hexasaccharide 26, the foregoing oligosaccharide was acetyl-
ated with acetic anhydride-pyridine to give 27.
Removal of the silyl protection by means of tetrabutyl-
ammonium fluoride (TBAF) afforded hexasaccharide 28, suitable
for the phosphate group introduction with N,N-diethyl-1,5-dihydro-
3H-2,3,4-benzodioxaphosphepin-3-amine and 1H-tetrazole, fol-
lowed by oxidation with m-chloroperbenzoic acid (m-CPBA),23
furnishing hexasaccharide 29 in 81% yield. A sharp peak in the
31P NMR spectrum (CDCl3, 162 MHz) at -0.36 ppm showed the
introduction of the protected phosphate, which was confirmed by
ESI MS (see the Supporting Information). Final deprotection was
performed in nearly quantitative yield by hydrogenation using flow
chemistry followed by mild Zemplen transesterification of the
acetyl esters. The structures of the purified hexasaccharides 1 and
2 were consistent with the native PS-II repeating unit,12 the main
difference being the presence of the linker connected to the
anomeric position of the mannosyl residue in the synthetic
molecules (see the Supporting Information).
In conclusion, the first synthesis of the C. difficile PS-II
hexasaccharide repeating unit has been successfully achieved.
In order to study the role of the phosphate group, both the
natural phosphate-containing hexasaccharide repeating unit
and its nonphosphorylated counterpart have been synthesized.
These compounds bear an R-O-linked aminopropyl spacer
at the anomeric position of the reducing end to allow their
conjugation to a carrier protein. Ongoing immunological
studies on the glycoconjugates prepared from the synthesized
molecules and the native polysaccharide will provide infor-
mation about the feasibility of a carbohydrate-based vaccine
against C. difficile.
(14) The moderate yield of 11 is due to the formation of variable amounts
of the orthoester which, besides rearranging to the desired product 11, was
partially hydrolyzed during the reaction course.
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Acknowledgment. We gratefully acknowledge Dr. A. Vivi
from NMR Center, University of Siena, for recording the
600 MHz NMR spectra.
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157.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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OL1026188
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