Synthesis and immunochemical evaluation of a non-methylated disaccharide analogue of the anthrax tetrasaccharide

Article abstract of DOI:10.1039/c2ob26131f

Anthrax tetrasaccharide is an oligosaccharide expressed at the outermost surface of the Bacillus anthracis spores, featuring three rhamnoses and a rare sugar called anthrose. This motif has now been identified as a plausible component of future human vaccines against anthrax. We report herein the synthesis of a 2-O-demethylated-β-d-anthropyranosyl-(1→3)-α-l- rhamnopyranose disaccharide analogue of this tetrasaccharide from a cyclic sulfate intermediate. This disaccharide conjugated to BSA induces an anti-native tetrasaccharide IgG antibody response when administered in BALB/c mice. Moreover, induced sera bound to native B. anthracis endospores. These results suggest that the disaccharide analogue, easily amenable for a synthetic scale-up, could be used in a glycoconjugate antigen formulation.

Full text of DOI:10.1039/c2ob26131f

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PAPER
Synthesis and immunochemical evaluation of a non-methylated disaccharide
analogue of the anthrax tetrasaccharide†
Ophélie Milhomme,a Susanne M. Köhler,b David Ropartz,c David Lesur,d Serge Pilard,d
Florence Djedaïni-Pilard,a Wolfgang Beyerb and Cyrille Grandjean*a,e
Received 13th June 2012, Accepted 24th August 2012
DOI: 10.1039/c2ob26131f
Anthrax tetrasaccharide is an oligosaccharide expressed at the outermost surface of the Bacillus anthracis
spores, featuring three rhamnoses and a rare sugar called anthrose. This motif has now been identified
as a plausible component of future human vaccines against anthrax. We report herein the synthesis of a
2-O-demethylated-β-D-anthropyranosyl-(1→3)-α-L-rhamnopyranose disaccharide analogue of this
tetrasaccharide from a cyclic sulfate intermediate. This disaccharide conjugated to BSA induces an
anti-native tetrasaccharide IgG antibody response when administered in BALB/c mice. Moreover, induced
sera bound to native B. anthracis endospores. These results suggest that the disaccharide analogue, easily
amenable for a synthetic scale-up, could be used in a glycoconjugate antigen formulation.
anthracis and related species,4 has made it possible to investigate
the development of glycoconjugate vaccines to replace or simply
Introduction
Bacillus anthracis, a Gram-positive, spore-forming soil bacter-
ium, is the etiologic agent of anthrax. Anthrax is primarily a zoo-
notic disease but because of the ease of production, storage, and
dissemination of the spores as well as the high lethality that
results from spore exposure (mainly by inhalation), this pathogen
has emerged as a potential biological warfare or bioterrorism
agent. B. anthracis strains are sensitive to antibiotics. However,
once commenced the treatment must be continued for prolonged
periods and is usually ineffective for inhaled anthrax which is
rarely recognized prior to the onset of bacteraemia and toxaemia.
Therefore, vaccination probably remains the best prophylactic
treatment.1,2
to complement the less than optimal currently licensed anthrax
vaccines (Fig. 1).
Seeberger et al. were the first to synthesize the tetrasaccharide
1 and to confirm its importance as an immune target.5 Further
antigenicity/immunogenicity studies have established that the
major antigenic determinants are expressed by the anthrose.
Although the rhamnose units of 1 are not essential,6–9 anti-
anthrax spore recognition consistently increases from anthrose to
the di/trisaccharide up to the tetrasaccharide.6,7 Focusing on
anthrose, the presence of a butanamido side-chain at C-4 seems
to be mandatory.7,10–12 Switching from a methyl to a hydroxy-
methyl group at C-6 leads to a complete loss of antigenicity,8
apparently owing to steric clash rather than modification of an
The disclosure of the structure of a tetrasaccharide 1,3 com-
prising a rare sugar called anthrose solely expressed by B.
aLaboratoire des Glucides, FRE CNRS 3517, Institut de Chimie de
Picardie, Université de Picardie Jules Verne, 33 rue Saint Leu, 80039
Amiens Cedex, France
bAnthrax-Laboratorium, Institut für Umwelt and Tierhygiene,
Universität Hoheheim Biogebaüde I, 1./2. Stockwerk, Garbenstrasse 30,
70599 Stuttgart, Germany
cINRA UR1268 BIA - Plate-forme IBiSA “Biopolymères, Biologie
Structurale” (BIBS), rue de la Géraudière, BP71627, 44316 Nantes
Cedex 3, France
dPlate-Forme Analytique, Université de Picardie Jules Verne rue
Dallery, 80039 Amiens Cedex, France
eUnité de Fonctionnalité et d’Ingénierie des Protéines, FRE CNRS
3478, UFR Sciences et Techniques, Université de Nantes, 2 rue de la
Houssinère, BP92208, 44322 Nantes Cedex 3, France.
E-mail: cyrille.grandjean@univ-nantes.fr; Fax: (+33) 251125632;
Tel: (+33) 251125732
1
†Electronic supplementary information (ESI) available: H and 13NMR
Fig. 1 Structure of the B. anthracis anthrose-terminated tetrasaccharide
antigen 1.
spectra of compounds 11, 13, 14 and 2. See DOI: 10.1039/c2ob26131f
8524 | Org. Biomol. Chem., 2012, 10, 8524–8532
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Scheme 1 Structures of di- and tetrasaccharide haptens related to the anthrax tetrasaccharide. Reagents and conditions: (a) 3-maleimidopropionic
acid N-hydroxysuccinimide ester, DIPEA, DMF, rt, 1 h, 38%; (b) biotinamidohexanoic acid N-hydroxysuccinimide ester, DIPEA, CH3CN, rt, 2 h, 86%.
important epitope as suggested by STD NMR experiments
carried out with a monoclonal anti-tetrasaccharide antibody and
a derivative related to 1.12 Noteworthy, replacement of the
methoxy at C-2 by a hydroxyl group does not impair the recog-
nition with anti-spores or anti-synthetic anthrax saccharide anti-
bodies.8,10 Moreover, serum induced in mice by conjugates
made of Shewanella capsular polysaccharides, which contain a
residue similar to anthrose but not methylated at O-2, have been
shown to cross-react with B. anthracis spores, suggesting further
that the methoxy at C-2 is not essential.11 From this set of data,
we have hypothesized that the truncated, demethylated anthro-
pyranosyl-rhamnopyranoside derivative 2 would be a valuable
mimic of the anthrax tetrasaccharide 1 while being more easily
accessible (Scheme 1).
On the one hand, reducing the length of the hapten from a
tetra- to a disaccharide would obviously result in a diminished
number of synthetic steps. On the other hand, whatever the start-
ing sugar or the strategy envisaged,5,8,10,13–22 extensive protect-
ing group manipulations are usually required to secure the
introduction of the 2-methoxy as in anthrose. Besides, this func-
tionalization must be carried out after the construction of the
β-glycosidic bond which links anthrose to the ultimate rhamnose
to avoid the formation of intractable α/β anomeric mixtures
during glycosylation.10,17
starting from D-fucose, its synthesis mainly consisting of introdu-
cing selectively the amide side-chain at C-4 according to an
SN2-reaction. To this end, we decided to apply our recently dis-
closed strategy which makes use of a 3,4-cyclic sulphate D-fuco-
pyranoside intermediate and which has led to the successful
preparation of both spacer-equipped disaccharide 3 and anthrax
tetrasaccharide 4 (Scheme 1).23,24
Thus, oxidation of known23 cyclic sulphite 5 under Sharpless
conditions gave rise to the corresponding cyclic sulphate 6
which was submitted to nucleophilic ring-opening using sodium
azide followed by aqueous acid hydrolysis of the resulting
acyclic sulphate to provide the 4-azido derivative 7 as the sole
regioisomer (50% yield for the 3 steps) (Scheme 2). Alterna-
tively, if 5 was first acetylated, 4-azido derivative 823 was
obtained in an improved 67% yield over the four steps. Further
acetolysis25 of 8 allowed the concomitant removal of the anome-
ric methyl group and protection of the remaining 3-OH to afford
the tri-acetylated intermediate 9. Selective deprotection of the
anomeric acetate using benzylamine and activation with
trichloroacetonitrile in the presence of DBU afforded glycoside
donor 11 (66% yield for the three steps). This intermediate was
thus obtained in 8 or 9 steps in 22 or 29% yield, respectively.
Glycosylation of donor 11 with known rhamnoside acceptor8
12 was next performed in the presence of TMSOTf to give disac-
charide 13 in 80% yield. Reduction of the azide in 13 was
carried out with sodium borohydride in the presence of nickel
chloride26 to provide an intermediate amine which was not iso-
lated but further acylated to introduce the anthrose amide side-
chain (55% for the two steps). Removal of the acetyl and
benzoyl groups of intermediate 14 under Zemplén conditions
followed by benzyl and Cbz-hydrogenolysis provided disacchar-
ide 2 (82% yield for the two steps).
We report herein the preparation of 2, its conjugation to BSA
used as a model protein carrier and the evaluation of the immune
response induced in mice by the conjugates compared to that
obtained by using the corresponding native disaccharide 3
(Scheme 1).
Results and discussion
Synthesis of the 2-O-demethylated-β-D-anthropyranosyl-(1→3)-
α-L-rhamnopyranose disaccharide 2
Preparation and characterization of the conjugates
Preparation of disaccharide 2 could be envisaged from a glycosy-
lation between a rhamnoside acceptor and an anthrose precursor
donor. The latter could, in turn, advantageously be obtained
Having the key disaccharide 2 and its native counterpart 323 for
comparison in hand, we next envisaged their conjugation to the
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Scheme 2 Synthetic route for the preparation of 2. Reagents and conditions: (a) NaIO4, RuCl3·H2O, CCl4–CH3CN–H2O (1 : 1 : 1.5), 0 °C, 4 h; (b)
NaN3, DMF, 60 °C, 3 h; (c) H2SO4, H2O, THF, rt, 1 h, 50% in three steps; (d) Ac2O–AcOH–H2SO4, rt, 6 h; (e) BnNH2, THF, rt, overnight; (f)
CCl3CN, DBU, CH2Cl2, 0 °C to rt, 2 h, 66% in three steps; (g) TMSOTf, 4 Å MS , CH2Cl2, −20 °C, 80%; (h) NaBH4, NiCl2·6H2O, EtOH–CH2Cl2,
rt, 1 h; (i) 3-hydroxy-3-methylbutanoic acid, HATU, DIPEA, CH2Cl2, rt, 18 h, 55%; ( j) MeONa (0.2 M in MeOH), rt, 12 h; (k) H2 (10 bars),
Pd(OH)2/C, 50 °C, 30 min, 82% in two steps.
BSA protein carrier. Both disaccharides 2 and 3 were coupled to
BSA using glutaraldehyde as a spacer via their respective free
amino groups. The conjugates were further stabilized through
reduction of the imine linkages to give conjugates 15 and 16
after dialysis (Scheme 3 and Table 1).
Alternatively, disaccharide 2 and BSA were derivatized using
a maleimide and an acetylthio cross-linker to give intermediates
17 (Scheme 1) and 18 (Scheme 3), respectively. Disaccharide 17
and protein 18 were conjugated in the presence of an excess of
hydroxylamine at pH 6.6 to afford the neoglycoconjugate 19
(Scheme 3 and Table 1).
We compared the MALDI-TOF mass spectra of the different
preparations with that of BSA to determine their extent of deriva-
tization (Table 1 and Fig. 2).
Similar molar sugar/protein ratios were found for conjugates
15 and 16 (19 and 18 mol mol−1, respectively) while that of 19
was significantly lower (6 mol mol−1 on a maximum of
12 mol mol−1 considering the extent of derivatization of 18).
Total carbohydrate content of preparation 19 was also deter-
mined using high pH high-performance anion-exchange chrom-
atography after TFA hydrolysis28 of the conjugate. The amount
of carbohydrate was very close to that estimated by mass spec-
trometry, suggesting that no or very little free sugar contaminant,
known to be sometimes associated with hyporesponsiveness,29,30
was present in vaccine preparation 19 (Table 1).
Scheme 3 Preparation of the conjugates.
Table 1 Characterization of the conjugates
Carbohydrate/protein
Conjugation
Conjugate chemistry
Yielda
(%)
mg/mg
μmol/μmol Ratio
15
16
19
Glutaraldehyde 78
Glutaraldehyde 85
Thio/maleimide 57
0.50/3.91
0.54/4.24
0.22/3.99 (0.20 0.36/0.06
HPAEC-PAD)
1.11/0.059 19
2.5/0.14 18
Immunogenicity of the glycoconjugates
6
To assess the immunogenicity of conjugates 15, 16 and 19,
groups of five female BALB/c mice were immunized sub-
cutaneously (sc) using an equivalent of 10 μg of disaccharide
per dose using the MPL® + TDM Adjuvant System (formerly
known as RIBI®) from Sigma. Each group received three doses
of the conjugates or phosphate buffered saline (PBS), used as a
control, at three-week intervals. Anti-tetrasaccharide antibody
(Ab) titers were determined by immobilizing biotinylated-
a Yields were calculated on the basis of the weight of protein in the
conjugate compared to the starting one determined by the Lowry
method.27
tetrasaccharide 20, obtained from 4 (Scheme 1), onto streptavi-
din-coated microtiter plates. PBS control sera contained low
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compares the IgG titers raised by the conjugates 15 and 16. No
statistical difference was observed between the Ab titers raised
against conjugates 15 and 19. However, further studies will be
needed to determine whether the immune response is influenced
by the conjugation chemistry since the two conjugates also differ
by their extent of hapten loading. Importantly, the sera obtained
after the third dose were shown to bind to B. anthracis spores
suggesting that they recognize the anthrax tetrasaccharide as
naturally expressed on exosporium (Fig. 5). These sera also
cross-reacted with B. cereus spores, known to possess the
anthrose biosynthetic operon4 (data not shown).
Finally, whatever the conjugate tested, the mean titre was
lower than that obtained when testing a hyperimmune serum
used as a positive control, suggesting that further B. anthracis
antigens should ideally be added to the carbohydrate hapten
valency in a glycoconjugate immunogen against anthrax.
In conclusion, the data presented herein reinforce the potential
use of synthetically accessible carbohydrate haptens to design a
glycoconjugate vaccine active against anthrax. In our hands,
demethylated disaccharide 2 proved to be as efficient an antigen
as its native counterpart 3, underlining that the 2-methyl group
of anthrose is not required for immunogenicity.
Experimental section
General remarks
All reactions were monitored by TLC on Kieselgel 60 F254
(E. Merck). Detection was achieved by charring with vanillin. Silica
gel (E. Merck, 240–400 mesh) was used for chromatography.
Optical rotation was measured with a JASCO DIP-370 digital
polarimeter, using a sodium lamp (λ = 589 nm) at 20 °C. All
NMR experiments were performed at 300.13 Hz using a Bruker
DMX300 spectrometer equipped with a Z-gradient unit for
pulsed-field gradient spectroscopy. Assignments were performed
by stepwise identification using COSY, successive RELAY,
HSQC and HMBC experiments using standard pulse programs
from the Bruker library. Chemical shifts are given relative to
external TMS with calibration involving the residual solvent
signals. For the chemical shift assignments of the disaccharides,
A and B refer to protons or carbons of the anthrose or the rham-
nose unit, respectively. Low-resolution ESI mass spectra were
obtained on a hybrid quadrupole/time-of-flight (Q-TOF) instru-
ment, equipped with a pneumatically assisted electrospray (Z-
spray) ion source (Micromass). High-resolution mass spectra
were recorded in positive mode on a ZabSpec TOF (Micromass,
UK) tandem hybrid mass spectrometer with EBETOF geometry.
The compounds were individually dissolved in MeOH at a con-
centration of 10 μg cm−3 and then infused into the electrospray
ion source at a flow rate of 10 mm3 min−1 at 60 °C. The mass
spectrometer was operated at 4 kV whilst scanning the magnet in
a typical range of 4000–100 Da. The mass spectra were collected
as continuum profile data. Accurate mass measurement was
achieved using polyethylene glycol as an internal reference with
a resolving power set to a minimum of 10 000 (10% valley).
Protein concentrations were measured with the Lowry
method27 using BSA as a standard. Sugar/protein ratios were
determined on an Autoflex III MALDI-TOF/TOF spectrometer
(Bruker Daltonics) in positive ionization modes and with a linear
Fig. 2 Positive linear ion mode MALDI-TOF MS spectra of BSA
(15 pmol) and of conjugates 15, 16, 18 and 19 (75 pmol). Mass spectra
were generated by averaging the signal of 2000 single laser shots.
levels of anti-tetrasaccharide Abs. In contrast, all conjugates eli-
cited anti-tetrasaccharide IgM and IgG Abs (Fig. 3).
Noteworthy, the mean IgG titres were maintained from the
second to the third immunization (the apparent increase was not
statistically significant). To further characterize the Ab response
induced by the conjugates, IgG-subclasses Ab responses to the
tetrasaccharide were measured in the sera obtained after the 3rd
immunization (Fig. 4). Sub-typing indicated a bias towards a
Th2 immune response as reflected by the prevalence of the IgG1
compared to the IgG2a,b response. A marked IgG3 response,
typical of an anti-carbohydrate response, was also punctually
observed. That conjugates of both native and 2-O-demethylated
disaccharides related to the upstream moiety of 1 could induce a
T-dependent immune response directed towards the anthrax tetra-
saccharide is clearly established or at least confirmed for the
former5b by these results. Previous antigenic8,10,12 and immune11
studies strongly supported that the presence of a methyl at O-2
of anthrose is not essential for the cross-reactivity. This new set
of data suggests that 2 is equivalent to 3 as a hapten if one
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Fig. 3 ELISA anti-anthrax tetrasaccharide antibody titres after two (a) and (c) and three (b) and (d) immunizations with PBS (open rhombus) and
conjugates 16 (square), 15 (triangle) and 19 (filled circle). ELISA plates were coated with biotinylated-derivative 20. Each data point represents the
titre for an individual mouse and the horizontal lines indicate the mean for the group of mice. A mouse of the group which had been immunized with
conjugate 19 died between the 2nd and the 3rd immunization. Since no death was observed among the control group, toxicity of conjugate 19 cannot
be ruled out. However, glycoconjugates are considered as safe and toxicity for anthrax tetrasaccharide-derived immunogens has not been reported pre-
viously. Moreover, the linker used to conjugate 2 to BSA is appropriate for use in humans.31 Comparison of all results from the immunized groups
with the control sera results were performed by ANOVA. *, P < 0.05; **, P < 0.01.
detection. 75 pmol of each samples were deposit on the MALDI
target plate and were mixed with sinapinic acid as the matrix
(10 mg mL−1; H2O–ACN–TFA, 50 : 50 : 0.1). The mass spec-
trometer was calibrated with a standard of BSA (15 pmol on
target) on dimer and different charge states of the protein.
RP-HPLC purifications were performed on a Prevail C18 (10
× 250 mm, 5 μm) column, at a 3 mL min−1 flow rate and a gradi-
ent of 0% B during 5 min, 0 to 100% solvent B over 25 min,
100% B, 5 min (eluent A: 0.05% TFA in H2O; eluent B: 0.05%
TFA in CH3CN–H2O 60 : 40).
Methyl 4-azido-4,6-dideoxy-α-D-glucopyranoside (7)
Fig. 4 Conjugates predominantly induce an anti-tetrasaccharide IgG1
To cyclic sulphite 523 (877 mg, 3.92 mmol) was added a cold
response. IgG subclasses were analyzed by ELISA using 20 as coating
agent (5 μg mL−1) and sera collected after the third dose at a 1/100
dilution. Each bar represents the sum of the IgG1, IgG2a, IgG2b and
IgG3 response for each individual from groups of mice immunized with
PBS (control), conjugate 16, 15 and 19, respectively.
solution of CCl4 (11.5 mL) and CH3CN (11.5 mL). The mixture
was cooled to 0 °C and cold water was added (17 mL).
RuCl3·H2O (13 mg, 0.06 mmol) and NaIO4 (1.68 g, 7.84 mmol)
were added in one portion and the reaction mixture was stirred
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reaction mixture was diluted with ethyl acetate and washed with
water and brine. The organic layer was dried (Na2SO4), filtered
and purified by flash chromatography using cyclohexane–EtOAc
(7 : 3) as eluent to give tri-acetylated intermediate 9. Rf 0.71
(cyclohexane–EtOAc 6 : 4); ESI-MS m/z 337.8 [M + Na]+. Part
of 9 (390 mg, 1.23 mmol) was further reacted with benzylamine
(135 μL, 1.23 mmol) in dry THF (5 mL) at room temperature
under inert atmosphere overnight and then quenched with
aqueous 1 N HCl. The solvent was evaporated under reduced
pressure and the crude residue was purified by flash chromato-
graphy using cyclohexane–ethyl acetate (6 : 4) as eluent to
provide 2,3 di-O-acetyl-4-azido-4,6-dideoxy-D-glucopyranose 10
(247 mg, 73%) as a colorless oil. Rf 0.45/0.35 (cyclohexane :
ethyl acetate 6 : 4); ESI-MS m/z 297.1 [M + Na]+. To a solution
of this intermediate (200 mg, 0.73 mmol) in CH2Cl2 (2 mL)
were added trichloroacetonitrile (1.62 mL, 16 mmol) and DBU
(25 μL, 0.16 mmol) dropwise at 0 °C. The mixture was then
stirred at room temperature for 2 h and then concentrated
in vacuo. The residue was purified by flash chromatography on
silica gel using 2% Et3N in cyclohexane–EtOAc (8 : 2) as eluent
to give trichloroacetimidate 11 (274 mg, 90%) as a pale yellow
oil as a mixture of α/β (>95/5) anomers. Rf 0.39 (cyclohexane–
Fig. 5 Accessibility of B. anthracis spore surface-exposed anthrax
tetrasaccharide by ELISA. Antibody titres after three immunizations
with PBS (open rhombus) (negative control) and conjugates 16 (square),
15 (triangle) and 19 (circle). ELISA plates were coated with spores from
B. anthracis A73 (Δ Ames) strain. Each data point represents the titre for
an individual mouse and the horizontal lines indicate the mean for the
group of mice. A positive control was provided by testing a hyper-
immune serum (see Experimental section for details). The comparisons
that are significantly different are PBS versus any group of conjugate-
immunized mice (P value < 0.05).
1
ethyl acetate 8 : 2); H NMR (300 MHz, CDCl3) δ 8.66 (s, 1 H,
NHα), 6.46 (d, 1 H, J1α,2α = 3.5 Hz, H-1α), 5.54 (t, 1 H, J2α,3α
J3α,4α = 10.1 Hz, H-3α), 5.06 (dd, 1 H, J1α,2α = 3.5 Hz, J2α,3α
10.1 Hz, H-2α), 3.99–3.87 (m, 1 H, H-5α), 3.34 (t, 1 H, J4α,5α
=
=
=
10.1 Hz, H-4α), 3.34 (t, 1 H, J4α,5α = 9.5 Hz, H-4α), 2.13 (s, 3
H, CH3), 2.01 (s, 3 H, CH3), 1.39 (d, 3 H, J5α,6α = 6.2 Hz, H-6
α); 13C NMR (75 MHz, CDCl3) δ 170.0 (CΟ), 169.6 (CΟ),
160.9 (CvNH), 93.1 (C-1α), 77.6 (CCl3 α), 70.8 (C-2α and
C-3α), 68.8 (C-5α), 65.5 (C-4α), 20.7 (CH3), 20.5 (CH3), 18.3
(C-6α); HR-ESIMS: m/z Calcd for C12H15Cl3N4O6 [M + Na]+:
438.9955. Found 438.9964.
vigorously at 0 °C. After 4 h stirring at this temperature, diethyl
ether was added and the layers were separated. The aqueous
layer was extracted with diethyl ether and the combined organic
layers were washed with brine, dried over MgSO4, filtered and
concentrated under vacuum to give crude sulphate 6. This
material is used in the next step without any further purification.
A mixture of crude 6 (940 mg, 3.92 mmol) and NaN3
(509 mg, 7.84 mmol) in dry DMF (5 mL) was stirred for 3 h at
60 °C. The solvent was then removed in vacuo. The residue was
suspended in dry THF (5 mL) and concd H2SO4 (205 μL,
3.92 mmol) and water (72 μL, 3.92 mmol) were added and the
mixture was stirred at room temperature for 1 h. The reaction
mixture was then quenched with NaHCO3 in excess and stirred
for 20 min. The mixture was then concentrated under vacuum
and the residue was purified by flash chromatography using
cyclohexane–EtOAc (1 : 1) as eluent to give azide 7 (592 mg,
50% over 3 steps) as a white solid. Rf 0.24 (cyclohexane–EtOAc
2-[N-(Benzyloxycarbonyl)amino]ethyl (2,3-di-O-acetyl-4-azido-
4,6-dideoxy-β-D-glucopyranosyl)-(1→3)-2-O-benzoyl-4-O-benzyl-
α-L-rhamnopyranoside (13)
Trimethylsilyl triflate (35 μL, 0.19 mmol) was added to a sol-
ution of donor 11 (200 mg, 0.48 mmol) and acceptor 12
(204 mg, 0.39 mmol) in CH2Cl2 (5 mL) containing 4 Å molecu-
lar sieves at −40 °C under an inert atmosphere. The temperature
of the reaction mixture was raised to −20 °C over 1 h and then
quenched upon addition of excess Et3N. The crude mixture was
filtered over a Celite® pad which was further washed with
CH2Cl2. The filtrate was next evaporated under reduced pressure
and the residue purified by flash chromatography on silica gel
using cyclohexane–ethyl acetate (7 : 3) as eluent to give disac-
charide 13 (241 mg, 80%). Rf 0.42 (cyclohexane–ethyl acetate
1
1 : 1); [α]D +154.5 (c 1, CHCl3). H NMR (300 MHz, CDCl3) δ
4.75 (d, 1 H, J1,2 = 3.9 Hz, H-1), 3.79 (t, 1 H, J2,3 = 9.5 Hz, J3,4
= 9.5 Hz, H-3), 3.64–3.56 (m, 2 H, H-2 and H-5), 3.43 (s, 3 H,
CH3), 3.07 (t, 1 H, J4,5 = 9.5 Hz, H-4), 1.34 (d, 3 H, J5,6 = 6.4
Hz, H-6); 13C NMR (75 MHz, CDCl3) δ 99.1 (C-1), 73.3 (C-3),
72.7 (C-2), 67.7 (C-4), 66.2 (C-5), 55.4 (CH3), 18.2 (C-6);
HR-ESIMS: m/z Calcd for C7H13N3O4 [M + Na]+: 226.0804.
Found 226.0800.
1
7 : 3); [α]D −2.5 (c 1, CHCl3). H NMR (300 MHz, CDCl3) δ
8.06 (br d, 2 H, J = 7.0 Hz, 2 H arom), 7.60 (br t, 1 H, J =
7.4 Hz, H arom), 7.51 (br t, 2 H, J = 7.4 Hz, H arom), 7.40–7.31
(m, 10 H, 10 H arom), 5.39 (dd, 1 H, J1B,2B = 1.8 Hz, J2B,3B
=
2,3-Di-O-acetyl-4-azido-4,6-dideoxy-D-glucopyranosyl
3.4 Hz, H-2B), 5.22 (br t, J = 5.8 Hz, NH), 5.16 (s, 2 H, CH2),
5.08 (t, 1 H, J2A,3A = J3A,4A = 9.6 Hz, H-3A), 4.95 (dd, 1 H,
J1A,2A = 7.9 Hz, J2A,3A = 9.6 Hz, H-2A), 4.89 (s, 1 H, H-1B),
4.87 (d, 1 H, J = 11.0 Hz, A part of an AB system), 4.77 (d,
1 H, J1A,2A = 7.9 Hz, H-1A), 4.65 (d, 1 H, J = 11.0 Hz, B part
trichloroacetimidate (11)
A solution of 8 (400 mg, 1.63 mmol) in Ac2O–AcOH–H2SO4
(2.15 : 0.37 : 0.015 mL) was stirred at room temperature for 6 h
and then was neutralized upon addition of NaHCO3 at 0 °C. The
This journal is © The Royal Society of Chemistry 2012
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of an AB system), 4.21 (dd, 1 H, J2B,3B = 3.4 Hz, J3B,4B = 9.2
Hz, H-3B), 3.86–3.74 (m, 2 H, H-5B and CHH), 3.60 (t, 1 H,
J3B,4B = J4B,5B = 9.9 Hz, H-4B), 3.64–3.55 (m, 1 H, CHH),
3.52–3.42 (m, 2 H, CH2), 3.39–3.31 (m, 1 H, H-5A), 3.19 (t, 1
H, J3A,4A = J4A,5A = 9.6 Hz, H-4A), 2.07 (s, 3 H, CH3), 1.77 (s,
3 H, CH3), 1.32 (d, 3 H, J5B,6B = 6.3 Hz, H-6B), 1.25 (d, 3 H,
J5A,6A = 6.1 Hz, H-6A); 13C NMR (75 MHz, CDCl3) δ 170.0
(CO), 169.7 (CO), 166.0 (CO), 156.4 (CONH), 138.0, 136.6,
133.1, 130.2, 129.9, 128.6, 128.5, 128.4, 128.2, 127.9, 127.8,
100.4 (C-1A), 97.3 (C-1B), 79.4 (C-4B), 78.8 (C3-B), 75.0
(CH2), 74.0 (C-3A), 72.2 (C-2B and C-2A), 70.6 (C-5A), 67.8
(C-5B), 67.1 and 66.9 (2 CH2), 65.4 (C-4A), 40.9 (CH2), 20.6
(CH3), 20.4 (CH3), 18.0 (C-6A and C-6B); HR-ESIMS: m/z
Calcd for C40H46N4O13 [M + Na]+: 813.3018. Found 813.2938.
(CH3), 17.0 and 16.6 (C-6A and C-6B); HR-ESIMS: m/z Calcd
for C45H56N2O15 [M + Na]+: 887.3578. Found 887.3589.
2-Aminoethyl 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-
α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside (2)
Compound 13 (67 mg, 0.08 mmol) was treated with NaOMe
(0.2 M solution in MeOH) (800 μL, 0.16 mmol, 2 equiv.) for
12 h at rt. The reaction mixture was then neutralized by resin
Amberlite IR120 H+. The resin was filtered off and the filtrate
was concentrated in vacuo. The crude product (54 mg,
0.08 mmol) was next treated without purification with a catalytic
amount of a Pd(OH)2/C under 10 bars of hydrogen at 50 °C for
30 min in EtOH/AcOH (9 mL/1 mL). The reaction mixture was
then concentrated in vacuo. The residue was dissolved in H2O
and was washed with CH2Cl2. The organic layer was extracted
by water. The aqueous phases were combined and concentrated
under reduced pressure. The crude was then purified by
RP-HPLC to give 2 (30 mg, 82% over 2 steps). [α]D = −7
2-[N-(Benzyloxycarbonyl)amino]ethyl [2,3-di-O-acetyl-4-(3-
hydroxy-3-methylbutanamido)-4,6-dideoxy-β-D-glucopyranosyl]-
(1→3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (14)
1
(CHCl3, c 0.5); H NMR (300 MHz, CDCl3) δ 4.79 (br s, 1 H,
H-1B), 4.64 (d, 1 H, J1A,2A = 7.7 Hz, H-1A), 4.13 (dd, 1 H,
J1B,2B = 1.7 Hz, J2B,3B = 3.3 Hz, H-2B), 3.95–3.87 (m, 1 H, A
part of an AB system, OCHH), 3.90 (dd, 1 H, J2B,3B = 3.3 Hz,
J3B,4B = 9.2 Hz, H-3B), 3.69–3.54 (d, 5 H, H-4A, H-5A, H-4B,
H-5B, B part of an AB system, OCHH), 3.47 (br t, 1 H, J =
9.4 Hz, H-3A), 3.35 (br t, 1 H, J = 8.4 Hz, H-2A), 3.27–3.15
(m, 2 H, CH2NH), 2.41 (s, NHCOCH2), 1.26 and 1.25 (2 × s, 2
CH3), 1.24 (d, 3 H, J5B,6B = 5.5 Hz, H-6B), 1.18 (d, 3 H, J5A,6A
= 5.7 Hz, H-6A); 13C NMR (75 MHz, CDCl3) δ 174.1 (CO),
103.4 (C-1A), 99.7 (C-1B), 79.3 (C-3B), 74.0 (C-2A), 73.2
(C-3A), 71.2 and 70.9 (C-4B and C-5B), 70.2 (C(CH3)2), 69.6
(C-2B), 68.6 (C-5A), 66.3 (OCH2), 56.5 (C-4A), 48.8 (CH2),
39.0 (CH2NH2), 28.2 and 28.1 (2 CH3), 17.1 (C-6B), 16.6
(C-6A); HR-ESI-MS m/z Calcd for C19H36N2O10 [M + H]+
453.2453, found 453.2428.
To a solution of azide 13 (149 mg, 0.18 mmol) in a mixture of
CH2Cl2 (2.8 mL) and EtOH (13 mL) was added NaBH4 (14 mg,
0.38 mmol) and a catalytic amount of NiCl2·6H2O. The reaction
mixture was stirred at room temperature for 1 h and then was
concentrated in vacuo. The residue was dissolved in CH2Cl2.
The organic layer was washed with water and brine, dried over
Na2SO4, filtered and concentrated in vacuo. The crude residue
was used in the next step without purification. Rf 0.1 (cyclo-
hexane–EtOAc 5 : 5); MS (ESI) m/z 787.1 [M + Na]+.
To a solution of crude amine in dry DMF (10 mL) was added
dropwise 3-hydroxy-3-methylbutanoic acid (45 μL, 0.36 mmol),
followed by HATU (142 mg, 0.36 mmol), and then DIPEA
(61 μL, 0.36 mmol). The reaction mixture was stirred under
argon at room temperature for 18 h and was then diluted with
CH2Cl2, washed with satd aq. NaHCO3 and water. The organic
phase was dried over Na2SO4, filtered and concentrated in
vacuo. The residue was purified by flash-chromatography on
silica gel using cyclohexane–EtOAc (6 : 4) as eluent to provide
intermediate 14 (90 mg, 55% over 2 steps) as a colorless oil. Rf
0.14 (cyclohexane–ethyl acetate 3 : 2); [α]D = −18.3 (c 0.5,
Conjugate 15
Disaccharide 2 (2 mg, 3.75 μmol, 50 equiv.) was added to a sol-
ution of BSA (5 mg, 0.075 μmol) in 20 mM potassium phos-
phate buffer solution, pH 6. The solution was cooled to 4 °C for
10 min and glutaraldehyde (10 μL, 50% in water) was added.
The reaction mixture was kept at 4 °C for 8 h and then NaBH4
was added. Following an additional period of 1 h at 4 °C, the
reaction mixture was extensively dialyzed against 0.1 M phos-
phate buffer, pH 7.5 and then freeze-dried.
1
CHCl3); H NMR (300 MHz, CD3OD) δ 7.94 (br dd, 2 H, J =
7.2 Hz, J = 1.5 Hz, 2 H arom), 7.57–7.50 (m, 1 H, H arom),
7.41 (br t, 2 H, J = 7.6 Hz, H arom), 7.32–7.16 (m, 10 H, 10 H
arom), 5.29 (br s, 1 H, H-2B), 5.06–4.92 (m, 2 H, H-3A and
CH2), 4.79–4.70 (m, 4 H, H-1A, H-2A, H-1B and A part of an
AB system), 4.55 (d, 1 H, J = 11.2 Hz, B part of an AB system),
4.17 (dd, 1 H, J2B,3B = 3.5 Hz, J3B,4B = 9.5 Hz, H-3B),
3.77–3.37 (m, 6 H, H-4A, H-5A, H-4B, H-5B and CH2),
3.31–3.23 (m, 2 H, CH2), 2.16 (s, CH2C(CH3)2OH), 1.81 (s, 3
H, CH3), 1.58 (s, 3 H, CH3), 1.14 (d, 3 H, J5B,6B = 6.2 Hz,
H-6B), 1.10 (s, 6 H, 2 × CH3), 0.98 (d, 3 H, J5A,6A = 6.0 Hz,
H-6A); 13C NMR (75 MHz, CD3OD) δ 172.8 (CO), 170.5 (CO),
169.9 (CO), 166.1 (CO), 157.6 (CONH), 138.3, 136.6, 133.0,
129.4, 128.2, 128.1, 128.0, 127.6, 127.4, 100.4 (C-1A), 97.0
(C-1B), 79.2 (C-4B), 78.7 (C3-B), 74.4 (CH2), 72.8, 72.6 and
72.5 (C-2A C-3A and C-2B), 70.5 (C-5A), 69.0 (C(CH3)2OH),
67.5 (C-5B), 66.3 and 66.1 (OCH2 and CH2Ph), 54.4 (C-4A),
48.1 (CH2), 40.3 (NCH2), 28.2 (2 CH3), 19.4 (CH3), 19.3
Conjugate 16
Disaccharide 3 (2 mg, 3.75 μmol, 50 equiv.) was added to a sol-
ution of BSA (5 mg, 0.075 μmol) in 20 mM potassium phos-
phate buffer solution, pH 6. The solution was cooled to 4 °C for
10 min and glutaraldehyde (10 μL, 50% in water) was added.
The reaction mixture was kept at 4 °C for 8 h and then NaBH4
was added. Following an additional period of 1 h at 4 °C, the
reaction mixture was extensively dialyzed against 0.1 M phos-
phate buffer, pH 7.5 and then freeze dried.
8530 | Org. Biomol. Chem., 2012, 10, 8524–8532
This journal is © The Royal Society of Chemistry 2012

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2-[N-(Maleimidopropionyl)amido]ethyl 4-(3-hydroxy-3-
methylbutanamido)-4,6-dideoxy-α-D-glucopyranosyl-(1→3)-
α-L-rhamnopyranoside (17)
to 500 mM. After every run, the column was re-equilibrated in
20 mM NaOH for 10 min. Analyses were carried out in
triplicates.
To a solution of disaccharide 2 (12 mg, 26 μmol) in DMF
(500 μL) were added 3-maleimidopropionic acid N-hydroxysuc-
cinimide ester (7 mg, 26 μmol, 1 equiv.) and DIPEA (9 μL,
52 μmol, 2 equiv.). The progress of the reaction was monitored
by HPLC. After 1 h at room temperature, the reaction was com-
plete. The crude was then purified by RP-HPLC to give 17
(6 mg, 38%) as a white solid after freeze-drying. ESI-MS m/z
625.9 [M + Na]+.
2-[N-(Biotinamidohexanoyl)amido]ethyl [4-(3-hydroxy-3-methyl-
butanamido)-4,6-dideoxy-2-O-methyl-β-D-glucopyranosyl]-
(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-
(1→2)-α-L-rhamnopyranoside (20)
To a solution of tetrasaccharide 4 (3 mg, 3.4 μmol) in acetonitrile
(100 μL) were added biotinamidohexanoic acid N-succinimidyl
ester (1.6 mg, 3.4 μmol, 1 equiv.) and DIPEA (0.9 μL, 5.2 μmol,
1.5 equiv.). The reaction mixture was stirred for 2 h at room
temperature and then concentrated in vacuo. The crude was then
purified by RP-HPLC to give 20 (3 mg, 86%) as a white solid
after lyophilisation. ESI-MS m/z 1120.6 [M + Na]+.
SATA-activated BSA 18
To a solution of BSA (7 mg, 0.105 μmol) dissolved in PBS
20 mM, pH 7.3 (700 μL), was added NHS-SATA (3 × 0.61 mg,
3 × 2.10 μmol, 3 × 20 equiv., dissolved in 30 μL of CH3CN) in
three portions every 45 min. The pH of the reaction mixture was
controlled (indicator paper) and maintained at 7–7.5 by addition
of 0.5 M aqueous NaOH. Following an additional reaction
period of 40 min, the crude reaction mixture was extensively
dialyzed against 0.02 M potassium phosphate buffer, pH 6.6
at 4 °C.
Immunizations
Groups of five 8-week-old female BALB/c mice were injected
subcutaneously three times at 3-week intervals with 10 μg of
conjugate, in saline solutions containing Sigma Adjuvant
System® (reference: S6322). The negative controlled group of
mice was immunized similarly with PBS. Mice were bled 1
week after the second and the third immunizations. The IgM and
IgG Ab response was measured by ELISA against the biotiny-
lated tetrasaccharide 20 immobilized on streptavidin-coated
microtiter plates as previously described.32,33 The Ab titre was
defined as the dilution of immune serum that gave an OD
(405 nm) at least twice that observed with pre-immune serum.
Conjugate 19
SATA-activated 18 in 20 mM potassium phosphate buffer sol-
ution was reacted with disaccharide 2 (1 mg, 1.60 μmol, 15.3
equiv.). Reaction mixture was buffered at a 0.5 M concentration
by addition of 1 M potassium phosphate buffer, pH 6.6. Then
NH2OH·HCl (7.5 μl of a 2 M solution in 1 M potassium phos-
phate buffer, pH 6.6) was added to the mixture and the coupling
was conducted for 2 h at room temperature. The conjugated
product was purified by dialysis against 0.1 M phosphate buffer,
pH 7.5, and freeze-dried.
Sugar content was determined both by MALDI-MS and by
high-performance anion-exchange chromatography following
TFA hydrolysis of the conjugate using disaccharide 3 as a stan-
dard28 as follows:
50 μL of sample was mixed with 12.5 μL of 10 N TFA (final
concentration 2 N) in a glass tube sealed with a screw-cap. After
2 h at 100 °C, solutions were freeze-dried and then re-dissolved
in 50 μl of water and the solutions transferred to autosampler
vials. In separate tubes, standard mixtures of known amounts of
disaccharide 3 (from 0 to 50 μg) were treated similarly and were
used for chemical identification and quantification. Chromato-
graphy of the samples was performed using a Dionex ICS 3000
system and a pulsed amperometric detector. 10 μL of the extract
were injected through a 4 × 50 mm Propac PA1 pre-column
(Dionex), before separation of anionic compounds on a 4 ×
250 mm Propac PA1 column (Dionex) at 35 °C, at a flow rate of
1 ml min−1. Peak analysis was performed using Chromeleon
software, version 7.0. Solvent A was H2O; solvent B was a
100 mM NaOH solution; and solvent C was a 100 mM NaOH
containing 0.5 M sodium acetate solution. Anionic compounds
were eluted with a multi-step gradient as follows: 0–7 min,
20 mM NaOH; 7–15 min, 20 to 100 mM NaOH; 15–60 min,
100 to 0 mM NaOH and simultaneous gradient of NaAc from 0
Anti-spore ELISA
If not stated otherwise, all incubation steps were carried out at
room temperature, alike all washing steps were carried out using
PBS containing 0.05% Tween 20. B. anthracis spores (A73)
were diluted in a 0.1 M carbonate buffer at a concentration of
108 spores per ml. Each well of a 96-well high binding microtiter
plate (Nunc, Roskilde, Denmark) was coated with 100 μl of the
prepared coating solution and incubated overnight at 4 °C.
Thereafter the wells were washed twice and blocked with 10%
(v/v) FCS in PBS for 1 h. Duplicates of the serum samples were
serial diluted in log2-steps in blocking solution on the plate at a
final volume of 100 μl per well and incubated for 2 h. As con-
trols a known positive serum (see below) (single serial dilution)
and a negative serum (single point measurement) and at least 6
blanks only containing blocking solution were included. After
washing the plates 5 times 100 μl of prepared secondary anti-
body solution in blocking solution was added to the wells and
incubated for 1 h. Antigen-specific antibodies used were poly-
clonal goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) con-
jugated to horseradish peroxidase. Plates were washed again 6
times and developed with 100 μl per well of 2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)
substrate (Roche Diagnostics, Manheim, Germany) and incu-
bated for 30 min in the dark. Absorbance was measured at
414 nm in a microtiter plate reader (Model 680, Biorad Labora-
tories, Hercules, CA). Endpoint titers were defined as the
This journal is © The Royal Society of Chemistry 2012
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Pilard, Org. Biomol. Chem., 2009, 7, 5184–5199.
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reciprocal of the highest serum dilution that resulted in an absor-
bance greater than an OD value of 0.1.
As a positive control, a hyperimmune sera prepared as follows
was used: Briefly, 10 NMRI mice were immunized with a
mixture of rBclA, rPA83, capsule-conjugate and formaldehyde-
inactivated spores including lipopeptide as an adjuvant. They
were immunized three times at two week intervals and were then
challenged with a 25 × LD50 of a virulent Ames strain. Three
weeks after the challenge the survivors were bled and killed. The
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Tukey’s post-test comparison. The results were significant if the
difference between the analysed groups equalled or exceeded the
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Acknowledgements
The authors are grateful to the Direction Générale de l’Arme-
ment and to the Deutsche Forschung Gesellschaft (DFG grant
BE 2157/3-) for financial support to O. M. and to S. M. K.,
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