Synthesis of a hexasaccharide repeating unit from bacillus anthracis vegetative cell walls

Article abstract of DOI:10.1021/ol7030262

The first synthesis of hexasaccharide 1 representing a repeat unit of a polysaccharide specific to the vegetative cell wall of Bacillus anthracis is reported. The synthetic hexasaccharide is equipped with an n-pentenyl handle at the reducing terminus to allow for further functionalization. Key transformations during the synthesis are the conversion of a glucose into a mannosazide residue, a (2+2) coupling, followed by double a-galactosylation to furnish the hexasaccharide, and global deprotection under Birch conditions.

Full text of DOI:10.1021/ol7030262

ORGANIC
LETTERS
2008
Vol. 10, No. 5
905-908
Synthesis of a Hexasaccharide
Repeating Unit from Bacillus anthracis
Vegetative Cell Walls
Matthias A. Oberli,† Pascal Bindscha
Peter H. Seeberger*
1
dler,† Daniel B. Werz,§ and
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH)
Zu¨rich, 8093 Zu¨rich, Switzerland
seeberger@org.chem.ethz.ch
Received December 17, 2007
ABSTRACT
The first synthesis of hexasaccharide 1 representing a repeat unit of a polysaccharide specific to the vegetative cell wall of Bacillus anthracis
is reported. The synthetic hexasaccharide is equipped with an n-pentenyl handle at the reducing terminus to allow for further functionalization.
Key transformations during the synthesis are the conversion of a glucose into a mannosazide residue, a (2+2) coupling, followed by double
r
-galactosylation to furnish the hexasaccharide, and global deprotection under Birch conditions.
Infections caused by the Gram-positive, spore-forming soil
bacterium Bacillus anthracis result in very serious disease.1,2
The durable form of the pathogen, the spores, is remark-
ably resistant to physical stress3 and has been used as a
biowarefare agent. Inhalation of spores that are ground into
fine particles that penetrate the lungs of people will kill
most victims if treatment does not commence within
24-48 h. The intentional release of anthrax spores through
contaminated letters caused the death of five people and
widespread panic among the U.S. population in autumn 2001.
Following these events, intensified efforts to detect B.
anthracis identified unique molecules on the surface of the
bacteria.
A unique tetrasaccharide 2 (Figure 1) on the surface of B.
anthracis spores was discovered4 and synthesized shortly
thereafter.5 Antibodies against this carbohydrate are able to
detect B. anthracis endospores in a highly selective manner.6
Carbohydrate-protein conjugates are currently evaluated in
challenge studies in animal experiments. Once inhaled,
anthrax spores germinate to produce vegetative cells within
30 min. An effective anthrax-subunit vaccine would likely
contain multiple antigens. Recently, another potential car-
(4) Daubenspeck, J. M.; Zeng, H.; Chen, P.; Dong, S.; Steichen, C. T.;
Krishna, N. R.; Pritchard, D. G.; Turnbough, C. L., Jr. J. Biol. Chem. 2004,
279, 30945-30953.
(5) (a) Werz, D. B.; Seeberger, P. H. Angew. Chem., Int. Ed. 2005, 44,
6315-6318. (b) Werz, D. B.; Adibekian, A.; Seeberger, P. H. Eur. J. Org.
Chem. 2007, 1976-1982. (c) Adamo, R.; Saksena, R.; Kova´cˇ, P. Carbohydr.
Res. 2005, 340, 2579-2582. (d) Adamo, R.; Saksena, R.; Kova´cˇ, P. HelV.
Chim. Acta 2006, 89, 1075-1089. (e) Mehta, A. S.; Saile, E.; Zhong, W.;
Buskas, T.; Carlson, R.; Kannenberg, E.; Reed, Y.; Quinn, C. P.; Boons,
G.-J. Chem. Eur. J. 2006, 12, 9136-9149. (f) Crich, D.; Vinogradova, O.
J. Org. Chem. 2007, 72, 6513-6520. (g) Guo, H.; O’Doherty, G. A. Angew.
Chem., Int. Ed. 2007, 46, 5206-5208.
† These authors contributed equally.
§ Present address: Institute for Organic and Biomolecular Chemistry,
University of Go¨ttingen, D-37077 Go¨ttingen, Germany.
(1) Mock, M.; Fouet, A. Annu. ReV. Microbiol. 2001, 55, 647-671.
(2) Sylvestre, P.; Couture-Tosi, E.; Mock, M. Mol. Microbiol. 2002, 45,
169-178.
(3) Nicholson, W. L.; Munakata, N.; Horneck, G.; Melosh, H. J.; Setlow,
P. Microbiol. Mol. Biol. ReV. 2000, 64, 548-572.
(6) Tamborrini, M.; Werz, D. B.; Frey, J.; Pluschke, G.; Seeberger, P.
H. Angew. Chem., Int. Ed. 2006, 45, 6581-6582.
10.1021/ol7030262 CCC: $40.75
© 2008 American Chemical Society
Published on Web 01/31/2008

Figure 1. Carbohydrate structures from Bacillus anthracis: The
spore surface tetrasaccharide 2 and the repeating hexasaccharide 3
of vegetative cell walls.
bohydrate antigen, the major cell wall polysaccharide 3, was
isolated from vegetative cells and structurally characterized
(Figure 1).7 This polysaccharide is also species-specific and
differs even from that of the closely related B. cereus strains.
The terminal repeating unit has not been described.7 How-
ever, any hexameric repeating unit itself constitutes an
attractive target for potential vaccine development and
pathogen detection.8
Here, we describe the first total synthesis of the hexa-
saccharide repeating unit 1 containing a terminal n-pentenyl
glycoside for subsequent functionalization and attachment
to a carrier protein.
Figure 2. Retrosynthetic analysis.
The doubly branched hexasaccharide 1 reveals several
challenging linkages such as two R-galactosidic linkages, an
R-linkage to a glucosamine residue and a â-linkage to a
mannosamine unit. Our retrosynthetic analysis of fully
protected hexasaccharide 4 (Figure 2) dissects the target
molecule into two terminal R-galactose building blocks 59
and two disaccharide parts A and B. For part A two building
blocks 6 and 8 are identified. The N-acetyl group of the
glucosamine of part A is masked as an azide to ensure
R-selectivity in the (2+2) glycosylation. The azide containing
building block 6 is derived from a precursor used in
carbohydrate antigen synthesis.10 Building block 8 is well-
known for the successful construction of terminal â-galactose
units.11 Trichloroacetyl-protected glucosamine 7 is used for
part B, and contains already the pentenyl handle that allows
for further transformations, into either an aldehyde6,12 or a
thiol.12a,13 The â-mannosamine linkage is introduced via
inversion of the C2-hydroxyl of glucose following construc-
tion of the â-glucosidic bond with 9.
The key carbohydrate subunits 6,10 7,14 and 915 were
readily prepared from known intermediates by using standard
protecting group transformations (Scheme 1). With these
monosaccharide building blocks in hand, the synthesis
commenced with the union of glucosamine n-pentenyl
glucoside 7 and glucose thioglycoside 9 to form a â(1f4)
glycosidic linkage.
The challenging â-mannosamine linkage was thus installed
by creating the trans-glucosidic linkage, readily prepared
with the help of the participating fluorenylmethoxycarbonate
(11) (a) Plante, O. J.; Andrade, R. B.; Seeberger, P. H. Org. Lett. 1999,
1, 211-214. (b) Hewitt, M. C.; Seeberger, P. H. Org. Lett. 2001, 3, 3699-
3702. (c) Wang, C.-C.; Lee, J.-C.; Luo, S.-Y.; Fan, H.-F.; Pai, C.-L.; Yang,
W.-C.; Lu, L.-D.; Hung, S.-C. Angew. Chem., Int. Ed. 2002, 41, 2360-
2362. (d) Werz, D. B.; Ranzinger, R.; Herget, S.; Adibekian, A.; von der
Lieth, C.-W.; Seeberger, P. H. ACS Chem. Biol. 2007, 2, 685-691.
(12) (a) Buskas, T.; So¨derberg, E.; Konradsson, P.; Fraser-Reid, B. J.
Org. Chem. 2000, 65, 958-963. (b) Wang, Z.-G.; Williams, L. J.; Zhang,
X.-F.; Zatorski, A.; Kudryashov, V.; Ragupathi, G.; Spassova, M.; Born-
mann, W.; Slovin, S. F.; Scher, H. I.; Livingston, P. O.; Lloyd, K. O.;
Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2719-2724.
(13) (a) Yamada, A.; Hatano, K.; Koyama, T.; Matsuoka, K.; Esumi,
Y.; Terunuma, D. Carbohydr. Res. 2006, 341, 467-473. (b) Isobe, H.; Cho,
K.; Solin, N.; Werz, D. B.; Seeberger, P. H.; Nakamura, E. Org. Lett. 2007,
9, 4611-4614.
(14) Melean, L. G.; Love, K. R.; Seeberger, P. H. Carbohydr. Res. 2002,
337, 1893-1916.
(15) (a) Zuurmond, H. M.; van der Klein, P. A. M.; van der Marel, G.
A.; van Boom, J. H. Tetrahedron Lett. 1992, 33, 2063-2066. (b) Vesely´,
J.; Rohlenova´, A.; Dzˇoganova´, M.; Trnka, T.; Tisˇlerova´, I.; Sˇaman, D.;
Ledvina, M. Synthesis 2006, 699-705.
(7) Choudhury, B.; Leoff, C.; Saile, E.; Wilkins, P.; Quinn, C. P.;
Kannenberg, E. L.; Carlson, R. W. J. Biol. Chem. 2006, 281, 27932-27941.
(8) Verez-Bencomo, V.; et al. Science 2004, 305, 522-525.
(9) (a) Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M. Org. Lett.
2003, 5, 987-989. (b) Tanaka, H.; Iwata, Y.; Takahashi, D.; Adachi, M.;
Takahashi, T. J. Am. Chem. Soc. 2005, 127, 1630-1631.
(10) (a) Garegg, P. J.; Konradsson, P.; Oscarson, S.; Ruda, K. Tetrahe-
dron 1997, 53, 17727-17734. (b) Turek, D.; Sundgren, A.; Lahmann, M.;
Oscarson, S. Org. Biomol. Chem. 2006, 4, 1236-1241.
906
Org. Lett., Vol. 10, No. 5, 2008

Differentially protected lactosamine building block 22 was
prepared from galactose phosphate 8 (Scheme 3). The
Scheme 1. Synthesis of 6, 7, and 9
Scheme 3. Synthesis of Disaccharide 22
pivaloyl (Piv) group as neighboring-participating group in
C2 ensured the selective creation of the â-linkage. Experi-
ments to use the thioglycoside 20 for direct glycosylation
with 19 proved unsuccessful. Thus, we converted the
Scheme 4. Assembly of Hexasaccharide 1
(Fmoc) group in C2.16 The Fmoc group was subsequently
cleaved and the C2 stereocenter was inverted via displace-
ment of the triflate by tetrabutylammonium azide to furnish
disaccharide 18 that contains the mannosamine motif.15b,17
Benzylidene protection of the C4 and C6 hydroxyls of the
glucose unit proved to be essential for the successful
inversion. Selective benzylidene ring opening yielded dis-
accharide acceptor 19 ready for glycosylation (Scheme 2).
Scheme 2. Synthesis of Disaccharide 19
Org. Lett., Vol. 10, No. 5, 2008
907

thioacetal via the hemiacetal 21 into glycosyl N-phenyltri-
fluoroacetimidate 22. This leaving group is a very attractive
alternative18 to the commonly used glycosyl trichloroacetimi-
dates.19 Glycosylation of disaccharide 19 with N-phenyltri-
fluoroacetimidate 22 in a mixture of dichloromethane and
diethyl ether afforded selectively tetrasaccharide 23 with the
corresponding R-glucosaminic linkage (Scheme 4).
To complete the synthesis, tetrasaccharide 23 was treated
with triethylamine to remove selectively the Fmoc group20
and afforded tetrasaccharide acceptor 24. This acceptor was
glycosylated with galactose building block 5 to yield 25.
Pentasaccharide 25 was treated with hydrazine monohydrate
to remove the levulinoyl ester. The final glycosylation with
galactose building block 5 afforded hexasaccharide 4.
Extensive spectroscopic investigations were performed to
identify the three diagnostic R-linkages, thereby confirming
the correct stereochemistry. Sodium in liquid ammonia
removed all permanent protecting groups and transformed
the azide moieties into amines. To afford NHAc groups the
completely deprotected carbohydrate was acetylated with a
mixture of acetic anhydride, pyridine, and DMAP. The
outcome of the Birch deprotection was shown to be highly
dependent on the neutralizing conditions: Using Amberlite
acidic resin we obtained low yields (15%) presumably since
the fully deprotected hexasaccharide stuck to the resin.
Employing acetic acid instead to neutralize resulted in greatly
improved yield (57%). Fragments from destructive fragmen-
tation could be observed as well. Saponification of 27 finally
yielded the desired target compound 1. Comparison of key
regions in the NMR spectra of 1 and 3 confirmed the
structural assignment of the isolated polysaccharide repeating
unit.21
In conclusion, we have reported a convergent total
synthesis of a Bacillus anthracis hexasaccharide repeating
unit of the major vegetative cell wall polysaccharide. The
target molecule was functionalized with a pentenyl handle
at the reducing terminus for conjugation to carrier proteins
or for attachment to microarrays. Our approach also allows
for facile synthesis of modifications and shorter sequences.
Immunological studies as well as the preparation of derivates
are under investigation. Results will be reported in due
course.
Acknowledgment. This work was supported by the Swiss
National Science Foundation (SNF grant 200020-109555)
and the ETH Zu¨rich. D.B.W. is grateful to the Deutsche
Forschungsgemeinschaft (DFG) for an Emmy Noether Fel-
lowship.
(16) (a) Grathwohl, M.; Schmidt, R. R. Synthesis 2001, 2263-2272. (b)
Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2004, 43, 602-605.
(17) (a) David, S.; Malleron, A.; Dini, C. Carbohydr. Res. 1989, 188,
193-200. (b) Classon, B.; Garegg, P. J.; Oscarson, S.; Tide´n, A.-K.
Carbohydr. Res. 1991, 216, 187-196.
(18) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405-2407.
(19) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19,
731-732.
(20) (a) Zhu, T.; Boons, G.-J. Tetrahedron: Asymmetry 2000, 11, 199-
205. (b) Roussel, F.; Takhi, M.; Schmidt, R. R. J. Org. Chem. 2001, 66,
8540-8548.
Supporting Information Available: Experimental pro-
cedures, spectroscopic data for all new compounds, and a
full citation for ref 8. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL7030262
(21) For detailed comparison of 1 and 3 see the Supporting Information.
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Org. Lett., Vol. 10, No. 5, 2008