Synthesis and antigenic analysis of the BclA glycoprotein oligosaccharide from the Bacillus anthracis exosporium

Article abstract of DOI:10.1002/chem.200601245

The glycoprotein BclA is an important constituent of the exosporium of Bacillus anthracis spores. This glycoprotein is substituted with an oligosaccharide composed of a β-L-rhamnoside substituted with the previously unknown terminal saccharide, 2-O-methyl

Full text of DOI:10.1002/chem.200601245

FULL PAPER
DOI: 10.1002/chem.200601245
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Synthesis and Antigenic Analysis of the BclA Glycoprotein Oligosaccharide
from the Bacillus anthracis Exosporium
Alok S. Mehta,[a, d] Elke Saile,[b, d] Wei Zhong,[a, d] Therese Buskas,[a] Russell Carlson,[a]
Elmar Kannenberg,[a] Yvonne Reed,[c] Conrad P. Quinn,*[b] and Geert-Jan Boons*[a]
Abstract: The glycoprotein BclA is an
important constituent of the exospori-
um of Bacillus anthracis spores. This
glycoprotein is substituted with an oli-
gosaccharide composed of a b-l-rham-
noside substituted with the previously
unknown terminal saccharide, 2-O-
methyl-4-(3-hydroxy-3-methylbutana-
mido)-4,6-dideoxy-d-glucopyranose,
also referred to as anthrose. Anthrose
has not been found in spores of B.
cereus and B. thuringiensis, making it a
potential species-specific marker for B.
anthracis. In order to study the antige-
nicity of anthrose, efficient syntheses of
an anthrose-containing trisaccharide
and a series of structurally related ana-
logues were developed. The analogues
lacked either the methyl ether at C-2
or contained modified C-4amino func-
tionalities of anthrose. The synthetic
compounds were equipped with an
aminopropyl spacer to facilitate conju-
gation to the carrier proteins maricul-
ture Keyhole Limpet Hemocyanin
(mcKLH) and bovine serum albumin
(BSA). Serum antibodies of rabbits im-
munized with live or irradiated spores
of B. anthracis Sterne 34F2 were able
to recognize the synthetic trisacchar-
ide–mcKLH conjugate. The specificity
of the interaction was confirmed by
competitive inhibition with the free-
and BSA-conjugated trisaccharides. In-
hibition using the trisaccharide ana-
logues demonstrated that the isovaleric
acid moiety of anthrose is an important
structural motif for antibody recogni-
tion. These data demonstrate that 1)
anthrose is a specific antigenic determi-
nant of the B. anthracis Sterne spore;
2) this antigen is presented to the
immune system of rabbits receiving the
anthrax live-spore vaccine; 3) synthetic
analogues of the oligosaccharide retain
the antigenic structure; and 4) the anti-
genic region is localized to specific ter-
minal groups of the oligosaccharide.
Collectively these data provide an im-
portant proof-of-concept step in the
synthesis and development of spore-
specific reagents for detection and tar-
geting of non-protein structures in B.
anthracis.
Keywords: anthrax
·
glycoconju-
gates · oligosaccharides · vaccines
[a] A. S. Mehta, W. Zhong, Dr. T. Buskas, Dr. R. Carlson,
Dr. E. Kannenberg, Dr. G.-J. Boons
Introduction
Complex Carbohydrate Research Center, University of Georgia
315 Riverbend Road, Athens, GA 30602 (USA)
Fax (+1)706-542-4412
Bacillus anthracis is a gram-positive, spore-forming bacteri-
um that causes anthrax in humans and other mammals.[1,2]
Because of the high resilience of Bacillus anthracis spores to
extremes of their environment they can persist for many
years until encountering a signal to germinate.[3,4] When
spores of B. anthracis are inhaled or ingested they may ger-
minate and establish populations of vegetative cells which
release anthrax toxins, often resulting in the death of the
host.[5] The relative ease by which B. anthracis may be
weaponized and the difficulty in early recognition of inhala-
tion anthrax due to the non-specific nature of its symptoms
were demonstrated by the deaths of five people who inhaled
spores from contaminated mail.[6–8] Consequently, considera-
ble efforts are being directed towards the development of
early disease diagnostics and there is a renewed interest in
anthrax vaccines. Sterile, cell-free vaccines containing the
E-mail: gjboons@ccrc.uga.edu
[b] Dr. E. Saile, Dr. C. P. Quinn
Centers for Disease Control and Prevention
Microbial Pathogenesis and Immune Response Laboratory
Meningitis and Special Pathogens Branch, NCID/DBMD
1600 Clifton Road, MS D-11, Atlanta, GA 30333 (USA)
Fax : (+1)404-639-2835
E-mail: cquinn@cdc.gov
[c] Y. Reed
Centers for Disease Control and Prevention
Scientific Resources Program, 1600 Clifton Road, MS D-11
Atlanta, GA 30333 (USA)
[d] A. S. Mehta, Dr. E. Saile, W. Zhong
These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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G.-J. Boons, C. P. Quinn et al.
protective antigen (PA) component of anthrax toxin have
proven safe and effective.[9,10] The anthrax vaccine that pro-
vides the most comprehensive protection is, however, the B.
anthracis Sterne 34F2 live-spore vaccine.[11,12] Although not
licensed for human use in the US or EU, the live-spore vac-
cine has proven highly efficacious as a veterinary vaccine
and similar live-spore preparations have been used exten-
sively in humans and animals in eastern Europe and Asia.[13]
Although these live-spore vaccines may elicit lower anti-
toxin antibodies than the licensed cell-free anthrax vaccines,
their documented efficacy is attributed to additional adju-
vant properties and as yet undefined protective epitopes
contributed by the spores or outgrowing vegetative cells.[14]
It is feasible, but as yet unexplored, that specific carbohy-
drate antigens may contribute to the enhanced efficacy of
the live spore vaccines.
Spores of B. anthracis are enclosed by a prominent loose
fitting layer called the exosporium, which consists of a para-
crystalline basal layer composed of a number of different
proteins and an external hair-like nap.[15–19] The filaments of
the nap are formed by the highly immunogenic glycoprotein
BclA, which has a long, central collagen-like region contain-
ing multiple X-X-Gly repeats where X can be any amino
acid.[20] Almost all of the repeating units contain a threonine
(Thr) residue, which provides sites for potential glycosyla-
tion.[21,22] Recently, it was shown that the BclA glycoprotein
contains an O-linked saccharide, the structure of which was
determined by a combination of NMR spectroscopy and
mass spectrometry.[23] The oligosaccharide is probably at-
tached to the protein through a GalNAc moiety, which was
lost during the hydrazine-mediated release from the BclA
glycoprotein.[23] The structure of the tetrasaccharide is de-
picted in Figure 1. The previously unknown non-reducing
terminal saccharide, 2-O-methyl-4-(3-hydroxy-3-methylbu-
tanmido)-4,6-dideoxy-d-glucopyranose, was named anthrose
and has not been found in spores of B. cereus and B. thurin-
giensis, making it a potential species-specific marker for B.
anthracis. It may also be a new target for therapeutic inter-
vention or vaccine development.[23]
logs. We demonstrate that 1) serum of rabbits immunized by
live or irradiated spores of B. anthracis Sterne 34F2 recog-
nize the trisaccharide 1, which is derived from the glycopro-
tein BclA; 2) the antigenic nature of the trisaccharide can
be altered by modification of specific side groups in the ter-
minal glycosyl structure; and 3) a 3-methyl butyryl substitu-
ent is essential for recognition by anti-spore antiserum.
Results and Discussion
Synthesis: To study the immunological properties of the oli-
gosaccharide of BclA, we examined whether antisera from
rabbits immunized with live or irradiated spores of B. an-
thracis Sterne 34F2 were able to recognize the synthetic an-
throse-containing BclA oligosaccharide[24–27] and selected an-
alogues. Although challenging, chemical synthesis offers an
opportunity to obtain almost every oligosaccharide target in
sufficient quantity and purity for these biological studies.
Furthermore, chemical synthesis has the advantage that a
target compound can be equipped with an artificial spacer
for convenient conjugation to a carrier protein, and offers
opportunities for obtaining analogues for structure–activity
relationship studies.
Compounds 1–4 (Figure 1) were selected as targets for
chemical synthesis. Compound 1 is derived from the oligo-
saccharide of BclA and contains an intact anthrose moiety.
Compound 2 lacks the methyl ether at C-2 and derivatives 3
and 4 contain modified C-4amino functionalities of an-
throse. We anticipated that compound 1 conjugated to
bovine serum albumin (BSA) or keyhole limpet hemocyanin
(KLH) would be attractive material for determining wheth-
er live or irradiated spores of B. anthracis Sterne 34F2 can
induce an anti-carbohydrate antibody response, and deriva-
tives 2–4 valuable to examine which chemical moieties of
anthrose are critical for binding with antibodies.
Compounds 1–4 were synthesized from monosaccharide
precursors 14, 15 and 9 or 13 (Schemes 1 and 2). Thus, gly-
cosyl donor 14[28] can be coupled with a benzyloxycarbonyl
protected amino propyl spacer to give compound 16, which
immediately can be used in a subsequent glycosylation with
rhamnoside 15 to give disaccharide 17. After removal of the
levulinoyl (Lev) ester of 17, the resulting glycosyl acceptor
can be coupled with an appropriately protected anthrose
donor. The benzoyl ester at C-2 of 15 will ensure that only
a-glycosides will be obtained during glycosylation due to
neighboring group participation.
In this paper, we report the synthesis of an anthrose-con-
taining trisaccharide and a series of structurally related ana-
The anthrose moieties of target compounds 1–4 are linked
through a b-glycoside to the C-3 hydroxyl of the rhamno-
side. Thus, an obvious strategy to introduce this moiety
would be the use of a glycosyl donor which carries a selec-
tively removable ester at C-2. At a late stage of the synthe-
sis, this protecting group can be removed to reveal an alco-
hol, which can then be methylated. However, this strategy is
complicated by the fact that the methylation has to be per-
formed under neutral or mildly acidic conditions due to the
presence of a number of base sensitive ester protecting
Figure 1. Oligosaccharide of glycoprotein BclA and synthetic targets.
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Anthrax Oligosaccharides
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trichloroacetimidate 9 by removal of the anomeric allyl
ether by treatment with PdCl2 and NaOAc followed by reac-
tion of the resulting lactol with trichloroacetonitrile in the
presence of 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU).[33,34]
G
Glycosyl donor 13 was synthesized from known thioglyco-
side 10.[35] Thus, a levulinoyl (Lev) ester at C-2 of compound
10 was installed by treatment with levulinic acid, 1,3-dicyclo-
hexylcarbodiimide (DCC), and 4-(dimethylamino)pyridine
(DMAP) in CH2Cl2 to give compound 11 in excellent
yield.[36] Next, the isopropylidene acetal of 11 was removed
by treatment with aqueous acetic acid to give the corre-
sponding diol. Attempts to selectively benzylate the C-3 hy-
droxyl of this compound by intermediate stannylene acetal
formation, using conditions described for the preparation of
7, gave 12 in a low yield due to cleavage of the Lev ester.
However, a moderate yield of 12 was obtained when the
stannene acetal formation was performed by refluxing the
diol and dibutyltin oxide in toluene followed by treatment
with benzyl bromide and tetrabutylammonium bromide
(Bu4NBr). Finally, triflation of 12 followed by nucleophilic
displacement with sodium azide gave the required thioglyco-
syl donor 13.
Next, attention was focused on the preparation of rham-
nosyl acceptor 18 and installment of the anthrose moiety.
Thus, an N-iodosuccinimide/trifluoromethanesulfonic acid
(NIS/TfOH) mediated glycosylation[37] of thioglycosyl donor
14 with benzyloxycarbonyl protected aminopropanol gave
spacer modified 16 as only the a-anomer. No self-condensa-
tion of 14 was observed due to a much higher glycosyl ac-
ceptor reactivity of N-benzyloxycarbonylaminopropanol.
Compound 16 was immediately used in a second glycosyla-
Scheme 1. a) MeI, NaH, DMF, RT; b) 1) 60% HOAc in H2O, 90 8C, 2)
Bu2SnO, MeOH, reflux, 3) CsF, BnBr, DMF, RT; c) 1) Tf2O, pyridine,
CH2Cl2, 08C, 2) NaN3, DMF, 408C; d) 1) PdCl2, NaOAc, 90% HOAc/
H2O, RT, 2) trichloroacetonitrile, DBU, CH2Cl2, RT; e) levulinic acid,
DCC, DMAP, CH2Cl2, RT; f) 1) 60% aq. HOAc, 908C, 2) Bu2SnO, tolu-
ene, reflux, 3) Bu4NBr, BnBr, toluene, reflux.
groups. In general, such procedures provide relatively low
yields of product, especially when applied to a complex
compound. Alternatively, the methyl ether can be intro-
duced at the monosaccharide stage by using strongly basic
conditions; however, this approach may suffer from the for-
mation of anomeric mixtures during the introduction of the
anthrose glycoside. In order to examine both strategies, gly-
cosyl donors 9 and 13 were prepared and coupled with gly-
cosyl acceptor 18. Compounds 9 and 13 contain an azido
moiety at C-4, which at a late stage of the synthesis can be
reduced to an amine and then acylated with different re-
agents to provide compounds 1–4.
Glycosyl donor 9 was synthesized from selectively protect-
ed allyl 6-deoxygalactoside 5 (Scheme 1).[29] Thus, methyla-
tion of the C-2 hydroxyl of 5
could easily be accomplished
by treatment of 5 with methyl
iodide in the presence of
sodium hydride to give com-
pound 6 in a yield of 99%.
The
3,4-O-isopropylidene
acetal of 6 could easily be re-
moved by using aqueous acetic
acid to give a diol, which was
selectively benzylated at C-3 to
give compound 7, by first stan-
nylene acetal formation by re-
action with dibutyltin oxide in
refluxing methanol followed by
treatment with benzyl bromide
and CsF in DMF.[30,31] Next, an
azido group was introduced at
C-4with inversion of configu-
ration to give compound 8 by
conversion of the hydroxyl of 7
into a triflate by reaction with
triflic anhydride and pyridine
followed by displacement with
sodium azide in DMF.[32] Fully
protected 8 was converted into
Scheme 2. a) HO(CH2)3NHZ, NIS, TfOH, CH2Cl2, 08C; b) levulinic acid, DCC, DMAP, CH2Cl2, RT; c)
A
NH2NH2·HOAc, MeOH, CH2Cl2, RT; d) 13, NIS, TfOH, CH2Cl2, 08C, 76%; e) 9, BF3·Et2O, MeCN, ꢀ408C,
86% a/b 1:4; f) MeI, Ag2O, Me2S, THF, RT; g) 1) 1,3-propanedithiol, TEA, pyridine, H2O, 2) for 22, 23, 24,
HOAt, HATU, DIPEA, RT, 61–76%, for 25, Ac2O, pyridine, RT, 22: 63%, 23: 78%, 24: 61%, 25: 66%; h) 1)
NaOMe, MeOH, RT, 2) Pd/C, H2 (g), tBuOH/H2O/AcOH 40:1:1, RT, 1: 98%, 2: 96%, 3: 94 %,4: 92%.
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G.-J. Boons, C. P. Quinn et al.
tion with glycosyl donor 15, using NIS/TfOH as the activator
to give disaccharide 17 in a good yield. Next, the levulinoyl
ester of 17 was selectively removed by treatment with hy-
drazine acetate,[36] to afford glycosyl acceptor 18 in a yield
of 93%. Coupling of trichloroacetimidate 9 with 18 in the
presence of BF3·Et2O in acetonitrile at ꢀ408C gave trisac-
charide 21 in a good yield (86%) as a 1:4mixture of a/b-
anomers. In this case, the modest b-selectivity was achieved
by the formation of an intermediate a-nitrilium ion.[38,39]
Anomerically pure 22 was obtained after reduction of the
azido group of 21 to give an amine, which was acylated with
3-hydroxy-3-methyl-butyric acid using O-(7-azabenzotriazol-
1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate/1-
hydroxy-7-azabenzotriazole/diisopropylethylamine (HATU/
HOAt/DIPEA) as the activating reagent.
As expected, an NIS/TfOH mediated coupling of thiogly-
cosyl donor 13 with acceptor 18 gave trisaccharide 19 as
only the b-anomer due to the neighboring group participat-
ing Lev ester at C-2. The Lev group of 19 was selectively re-
moved by treatment with hydrazine acetate[36] and the hy-
droxyl of the resulting trisaccharide 20 was methylated by
treatment with methyl iodide and freshly prepared Ag2O in
the presence of dimethyl sulfide. Despite a prolonged reac-
tion time, the product was obtained in a modest yield of
51%. Thus, the advantage of using glycosyl donor 13 in tri-
saccharide formation was off-set by a low yielding methyla-
tion reaction.
Reduction of the C-4’’ azido moiety of 21 followed by the
coupling with 3-hydroxy-3-methyl-butyric acid gave com-
pound 22. Deprotection of 22 could easily be accomplished
by a two-step procedure entailing removal of the benzoyl
esters using sodium methoxide in methanol, followed by
cleavage of the benzyl ethers and benzyloxycarbamate by
hydrogenation over Pd/C in a mixture of tert-butanol/water/
acetic acid.
Analogue 2, lacking a methyl ether at C-2 of anthrose,
was prepared by reduction of the azido group of 20 followed
by introduction of the 3-hydroxy-3-methyl-butyric acid
moiety and deprotection using standard procedures. Com-
pounds 3 and 4 were obtained by reduction of the azido
moiety of 21 followed by acylation of the resulting amine
using appropriate reagents to give compounds 24 and 25,
which were deprotected using standard procedures.
mido) propionate (SBAP) in a sodium phosphate buffer
(pH 7.2) containing 0.15m sodium chloride and then purified
by a centrifugal filter device with a nominal molecular-
weight limit of 30 kDa. The bromoacetyl activated KLH
(KLH-BrAc) was subsequently incubated with the thiolated
trisaccharide in a 0.1 mm sodium phosphate buffer (pH 8.0)
containing 5 mm ethylene diamine tetraacetate (EDTA).
The afforded glycoconjugate (KLH-BrAc-1) carried 1042
copies of trisaccharide 1 per KLH molecule as determined
by Lowryꢁs protein concentration test and quantitative car-
bohydrate analysis by HPAEC-PAD. For the purpose of
evaluating the binding specificity of antibodies raised
against the B. anthracis spores, the thiol derivative of trisac-
charide 1 was conjugated to maleimide activated BSA
(BSA-MI, Pierce Endogen, Inc.) in a phosphate buffer
(pH 7.2) containing sodium azide and EDTA. After a reac-
tion time of 2 h, the glycoprotein was purified using a centri-
fugal filter device with a nominal molecular weight cut-off
of 10 kDa. The average number of trisaccharide copies per
BSA molecule was determined to be 18:1. The same conju-
gation method and thiolated derivatives of trisaccharides 2,
3, and 4 were used to give the corresponding BSA-MI-2,
BSA-MI-3, and BSA-MI-4 glycoconjugates with a sacchride/
protein ratio of 10:1, 9:1, and 4:1, respectively.
Antibody binding analyses: To explore the immunogenicity
of the saccharide moieties of BclA, rabbits were immunized
four times at biweekly intervals with live or irradiated
spores of B. anthracis Sterne 34F2. First, it was investigated
whether the post-immune sera have the ability to recognize
the synthetic anthrose-containing trisaccharide 1. For this
purpose, an ELISA was performed whereby microtiter
plates were coated with the KLH-BrAc-1 conjugate and
serial dilutions of sera added. An anti-rabbit IgG antibody
labeled with horseradish peroxidase was employed as a sec-
ondary antibody for colorimetric detection (OD, optical
density). Binding was observed between the antisera and
KLH–trisaccharide conjugate whereas no interaction was
detected for native KLH, indicating that the saccharide epit-
opes of BclA are antigenic (Figure 2a). Rabbits immunized
with irradiated Sterne34F2 spores elicited lower but detecta-
ble titers of anti-saccharide antibodies. The fact that the ir-
radiated spores elicited IgG antibodies indicates that the
saccharide epitopes were not damaged during the irradiation
process.
Next, the specificity of the interaction of the antisera with
the KLH-BrAc-1 conjugate was further investigated using a
competitive inhibition ELISA. Thus, microtiter plates were
coated with the KLH-BrAc-1 conjugate, and serial dilutions
of antisera mixed with free trisaccharide 1 were added. As
depicted in Figure 2b, a six-fold excess of trisaccharide 1 (as
compared to a concentration of trisaccharide used for coat-
ing microtiter wells), resulted in a significant drop in OD at
all serum dilutions tested. Also, increasing the excess of the
competing trisaccharide 1 resulted in a further reduction in
OD. It is evident that the inhibition is dose dependent, thus
demonstrating that the interaction of the elicited antibodies
Preparation of carbohydrate–protein conjugates: Trisacchar-
ide 1 was linked to the carrier protein mariculture Keyhole
Limpet Hemocyanin (mcKLH; Pierce Biotechnology, Rock-
ford, IL) for immunological evaluation. To this end, the
amino functionality of trisaccharide 1 was derivatized with
an acetyl thioacetic acid moiety by reaction with S-acetylth-
ioglycolic acid pentafluorophenyl ester to afford the corre-
sponding thioacetate derivative, which after purification by
size-exclusion chromatography, was directly de-S-acetylated
using 7% ammonia (g) in DMF just prior to conjugation.
The de-S-acylation was performed under a strict argon at-
mosphere to prevent formation of the corresponding disul-
fide. KLH was activated with succinimidyl 3-(bromoaceta-
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Figure 3. Competitive inhibition of anti-live spore antiserum binding to
synthetic anthrose-containing trisaccharide by synthetic analogue conju-
gates. Microtiter plates were coated with KLH-BrAc-1 conjugate (0.5 mg
per mL conjugate corresponding to 0.03 mg per mL trisaccharide). Rabbit
anti-live spore B. anthracis Sterne 34F2 antiserum (1:1600 dilute) was first
mixed with BSA–trisaccharide conjugates (0–128-fold excess, wt/wt based
on carbohydrate concentration) and then applied to the coated microtiter
plate. Unconjugated BSA mixed with antiserum did not have any effect
(data not shown). OD values were normalized for the OD values ob-
tained without BSA-trisaccharide conjugate (0-fold “excess”, 100%).
Non-specific binding was tested with uncoated wells containing antiserum
and buffer (data not shown). The data are reported as the means ꢁSD
of triplicate measurements.
ide analogue 2, which lacks the 2’’-O-methyl ether but has
an intact N-(3-hydroxy-3-methyl-butyryl) moiety at C-4’’. As
shown in Figure 3, this conjugate is a potent inhibitor of an-
tibody binding with as low as a 2-fold weight excess eliciting
>95% reduction in reporter signal, compared to the BSA-
MI-conjugate carrying the native trisaccharide 1, for which
no significant difference in inhibition was observed in the
concentration range investigated. These data indicate that
the methyl ether is not critical for anti-spore antibody bind-
ing. To elucidate the importance of the 3-hydroxy-3-methyl-
butyryl moiety of anthrose, conjugates BSA-MI-3 and BSA-
MI-4 were prepared. Trisaccharide 3 carries a 3-methyl-bu-
tyryl moiety at the C-4’’, thus only lacking the hydroxyl
group of the native C-4-moiety of the anthrose monosac-
charide, whereas trisaccharide 4 is N-acetylated at the C-4’’,
thus lacking most of the 3-hydroxy-3-methyl-butyryl moiety.
Interestingly, a two-fold excess of trisaccharide 3 reduced
OD by 85% compared to the control. In contrast, a similar
concentration of analogue 4 resulted in reduction in OD of
only 17%. Very high concentrations of BSA-MI-4 were re-
quired to achieve considerable inhibition (a 500-fold excess
of BSA-MI-4 resulted in a 50% drop in OD, data not
shown). These results indicate that the 4’’-(3-methylbutyryl)-
moiety is an important structural motif of the authentic sac-
charide epitope on the surface of B. anthracis Sterne spores.
Figure 2. ELISA and competitive inhibition of anti-live and anti-irradiat-
ed spore antiserum. Microtiter plates were coated with KLH-BrAc-1 con-
jugate (0.5 mg per mL conjugate, corresponding to 0.03 mg per mL trisac-
charide). Rabbit anti-live (1:200!1:6400 diluted) or anti-irradiated
(1:10!1:3000 diluted) spore B. antracis Sterne 34F2 antisera were applied
to coated microtiterplates (a). For the inhibition assay the serum was first
mixed with free trisaccharide 1 (0–200-fold excess, wt/wt) (b and c). Un-
specific binding was tested with uncoated wells with 200-fold “excess” tri-
saccharide or 200-fold “excess” KLH (data not shown). The data are re-
ported as the means ꢁSD of triplicate measurements.
with 1 is specific. The interaction of antisera from rabbits
immunized with irradiated spores with 1 could also be inhib-
ited in a dose response manner (Figure 2c).
Having established that Sterne 34F2 spores are able to
induce an anti-carbohydrate antibody response, we sought
to further evaluate which structural motifs of the anthrose
moiety are critical for antibody recognition. To this end, the
ability of BSA-MI-1 and BSA-MI-conjugates of the three
structural analogues 2, 3, and 4 to inhibit the interaction of
the antisera with KLH-BrAc-1 was determined (Figure 3).
For these experiments, BSA conjugates were employed in
an effort to conserve synthetic material. Microtiter plates
were again coated with the KLH-BrAc-1 conjugate and
treated with an antisera dilution of 1:1600. The importance
of the 2’’-O-methyl ether of anthrose was established using
the BSA-MI-2 conjugate. This conjugate carries trisacchar-
Conclusion
The significance of the observations described in this paper
is two-fold. First, we have demonstrated that, by using anti-
live spore antisera and anti-irradiated spore antisera, the an-
throse-containing trisaccharide of BclA is antigenic and ex-
posed on the surface of B. anthracis Sterne 34F2 spores
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G.-J. Boons, C. P. Quinn et al.
trated to dryness. The residue was co-distilled with toluene twice. Purifi-
cation of the crude product by column chromatography on silica gel af-
forded the desired diol product.
when presented in rabbits. Second, we have located an im-
portant antigenic component of this reactivity in the termi-
nal 3-methyl-butyryl structures of the saccharide and con-
firmed its specificity using synthetic saccharide analogues.
These data provide an important proof-of-concept step in
the development of spore-specific reagents for detection and
targeting of non-protein structures in B. anthracis. These
structures may in turn provide a foundation for directing
immune responses to spore structures during the early
stages of the B. anthracis infection process. During the prep-
aration of this manuscript Seeberger and co-workers report-
ed that the anthrax oligosaccharide conjugated to KLH
could elicit antibodies that recognize B. anthracis spores.[40]
Our data are complementary to these findings in that B. an-
thracis spores elicit anti-carbohydrate antibodies, which may
be harnessed for diagnosis. Ongoing studies will demon-
strate whether these and additional saccharide structures are
present and accessible on the spores from other B. anthracis
isolates, including the highly virulent B. anthracis Ames and
other B. anthracis cured of virulence plasmids pXO1 and
pXO2.
General procedure for introduction of the C-4 azide group: Trifluorome-
thanesulfonic anhydride (1.5 equiv) was added slowly at 08C to a solution
of 7 or 12 (1 equiv) in pyridine (10 equiv) and dry CH2Cl2 (at a concen-
tration of 0.2 mol saccharide per L). The reaction mixture was stirred at
08C for 1 h, and then diluted with CH2Cl2. The solution was washed with
H2O and saturated NaHCO3. The organic layer was dried (MgSO4), fil-
tered, and concentrated to dryness. To a solution of this residue in DMF
(at a concentration of 0.08 mol saccharide per L) was added sodium
azide (5 equiv). The reaction mixture was stirred at 408C overnight, and
then concentrated to dryness. The residue was dissolved in ethyl acetate,
and the solution was washed with saturated NaHCO3 and brine. The or-
ganic layer was dried (MgSO4), filtered, and concentrated to dryness. Pu-
rification of the crude product by column chromatography on silica gel
afforded the desired product 8 or 13.
General procedure for cleavage of the levulinoyl ester: A solution of hy-
drazine acetate (1 equiv) in dry MeOH (0.4molL ꢀ1) was added under
argon to a solution of 17 or 19 (1 equiv) in dry CH2Cl2 (at a concentra-
tion of 0.04mol saccharide per L). The reaction mixture was stirred at
room temperature for 4h, and then concentrated to dryness. The residue
was dissolved in CH2Cl2, and then washed with water. The organic layer
was dried (MgSO4), filtered, and concentrated to dryness. Purification of
the crude product by column chromatography on silica gel afforded the
desired product 18 or 20.
General procedure for azide reduction and introduction of C-4’’ moiety:
TEA (15 equiv) was added to a solution of 20 or 21 (1 equiv) and 1,3-
propanedithiol (20 equiv) in pyridine (at a concentration of 0.014mol
saccharide per L) and H2O (at a concentration of 0.1 mol saccharide per
L). The reaction mixture was stirred at room temperature overnight, and
then concentrated to dryness. The residue was co-evaporated with tolu-
ene twice and ethanol twice. Purification of the crude product by column
chromatography on silica gel (CH2Cl2/MeOH/TEA 100:5:1) afforded the
free amine compounds. b-Hydroxyisovaleric acid or isovaleric acid
(2 equiv) was activated by HOAt (4equiv) and HATU (4equiv) in DMF
(at a concentration of 0.01 mol saccharide per L) for 1 h, and then
DIPEA (8 equiv)was added. The resulting yellow solution was added
dropwise to the free amine compound (1 equiv) in DMF (at a concentra-
tion of 0.02 mol saccharide per L). The reaction mixture was stirred at
room temperature for 4h, and then concentrated to dryness. Purification
of the crude product by column chromatography on silica gel afforded
the desired product 22, 23 or 24. Alternatively, a solution of free amine
(1 equiv) in Ac2O (2 equiv), pyridine (2 equiv) and DMAP (0.1 equiv)
was stirred at room temperature overnight, and then concentrated to dry-
ness. The residue was co-evaporated with toluene twice. Purification of
the crude product by column chromatography on silica gel afforded the
desired product 25.
Experimental Section
General: 1H NMR spectra were recorded in CDCl3 or D2O on Varian
Merc-300 or Varian Inova-500 spectrometers equipped with Sun worksta-
tions at 300 K. TMS (dH 0.00) or D2O (dH 4.67) was used as the internal
reference. 13C NMR spectra were recorded in CDCl3 or D2O at 75 MHz
on a Varian Merc-300 spectrometer, respectively, using the central reso-
nance of CDCl3 (dC 77.0) as the internal reference. COSY, HSQC,
HMBC and TOCSY experiments were used to assist signal assignment of
the spectra. The different monosaccharide units are referred to as a, b,
and c, respectively, with a denoting the reducing end monosaccharide.
Mass spectra were obtained on Applied Biosystems Voyager DE-Pro
MALDI-TOF (no calibration) and Bruker Daltonics 9.4T (FTICR, exter-
nal calibration with BSA). Optical rotatory power was obtained on Jasco
P-1020 polarimeter at 300 K.
Chemicals were purchased from Aldrich or Fluka and used without fur-
ther purification. CH2Cl2, acetonitrile and toluene were distilled from cal-
cium hydride; THF from sodium; and MeOH from magnesium and
iodine. Mariculture keyhole limpet hemocyanin (mcKLH), maleimide ac-
tivated bovine serum albumin (BSA-MI), and succinimidyl 3-(bromoace-
tamido)propionate (SBAP) were purchased from Pierce Endogen, Rock-
ford, IL. Aqueous solutions are saturated unless otherwise specified. Mo-
lecular sieves were activated at 3508C for 3 h in vacuo. All reactions
were performed under anhydrous conditions under argon and monitored
by TLC on Kieselgel 60 F254(Merck). Detection was by examination
under UV light (254nm) and by charring with 10% sulfuric acid in meth-
anol. Silica gel (Merck, 70–230 mesh) was used for chromatographies. Ia-
trobeads 6RS-8060 was purchased from Bioscan.
General precedure for global deprotection: NaOMe (pH 8–10) was
added to a solution of 22, 23, 24 or 25 in dry MeOH (at a concentration
of 0.06 mol saccharide per L). The reaction mixture was stirred at room
temperature overnight, and then neutralized by the addition of Dowex
650 H+. The suspension was filtered through Celite, and washed with
MeOH/CH2Cl2 1:1. The combined filtrates were concentrated to dryness.
Purification of the crude product by column chromatography on silica gel
afforded the desired deacetylated product. To a solution of the partially
deprotected compound in tert-butanol/H2O/AcOH (40:1:1, 0.01 molLꢀ1
)
General procedure for levulination: A solution of DCC (6 equiv) and
DMAP (0.015 equiv) in CH2Cl2 was added under argon to a solution of
10 or 14 (1 equiv) and levulinic acid (10 equiv) in CH2Cl2 (at a concentra-
tion of 0.06 mol saccharide per L). The reaction mixture was stirred at
room temperature for 2 h, and then filtered through Celite. The filtrate
was washed twice with water. The organic layer was dried (MgSO4), fil-
tered, and concentrated to dryness. Purification of the crude product by
column chromatography on silica gel afforded the desired product 11 or
15.
was added Pd/C (cat.) under an atmosphere of hydrogen. The reaction
mixture was stirred at room temperature overnight, and then filtered
through Celite. The filtrate was concentrated to dryness. Purification of
the crude product by Iatro beads afforded the desired product 1–4.
Allyl 3,4-O-isopropylidene-2-O-methyl-a-d-fucopyranoside (6): NaH
(3.25 g, 67.63 mmol, 50% in mineral oil) was added to a solution of 5
(8.26 g, 33.81 mmol) in DMF (90 mL). The reaction mixture was stirred
at 08C for 1 h, and then methyl iodide (4.21 mL, 67.62 mmol) was added
dropwise. The reaction mixture was stirred at room temperature for 6 h,
and then poured into ice water. The solution was extracted with CH2Cl2
(100 mL) and washed with water (100 mL). The organic layer was dried
General procedure for isopropylidene removal: A solution of 6 or 11
(1 equiv) in acetic acid/water (3:2, at a concentration of 0.5 mol saccha-
ride per L) was heated under reflux at 908C for 15 min, and then concen-
9142
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9136 – 9149

Anthrax Oligosaccharides
FULL PAPER
(MgSO4), filtered, and concentrated to dryness. Purification of the crude
product by column chromatography on silica gel (hexane/EtOAc 4:1) af-
forded the desired product 6 as colorless oil (8.66 g, 99%). Rf =0.74
(hexane/EtOAc 2:1); [a]2D7 =+67.7 (c=3.6 in CHCl3); 1H NMR
(300 MHz, CDCl3): d = 1.26 (d, 3H, J5,6 =6.3 Hz, H-6), [1.29, 1.47, 2ꢂs
(CH3CCH3)], 3.30 (dd, 1H, J1,2 =3.6, J2,3 =8.1 Hz, H-2), 3.44 (s, 3H,
OCH3), 3.94–4.10 (m, 3H, H-4, H-5, OCH2CHCH2), 4.14 (dd, 1H, J=
5.4, 12.9 Hz, OCH’2CHCH2), 4.18 (dd, 1H, J2,3 =8.1, J3,4 =5.7 Hz, H-3),
(m, 1H, OCH2CHCH2), 7.19–7.35 (m, 5H, Harom); 13C NMR (75 MHz,
CDCl3): d
(OCH2CHCH2), 75.5 (PhCH2), 79.8 (C-3), 82.3 (C-2), 94.8 (C-1), 118.3
(OCH2CHCH2), [127.8, 128.2, 128.4, 133.6 (Carom)], 138.2
= 18.4(C-6), 58.7 (OCH 3), 66.1 (C-5), 67.9 (C-4), 68.2
(OCH2CHCH2); MALDI-TOF MS: m/z: calcd for C17H23N3O4Na:
356.16; found: 356.7 [M+Na]+.
Ethyl
3,4-O-isopropylidene-2-O-levulinoyl-1-thio-b-d-fucopyranoside
(11): Treatment of 10 (1.34g, 5.40 mmol) and levulinic acid (5.53 mL,
54.00 mmol) in CH2Cl2 (90 mL) with DCC (6.69 g, 32.42 mmol) and
DMAP (9.90 mg, 0.081 mmol) in CH2Cl2 (9 mL) according to the general
procedure gave compound 11 as colorless oil (1.76 g, 94%). Rf =
0.71(hexane/EtOAc 1:1); [a]2D7 =+1.3 (c=0.7 in CHCl3); 1H NMR
(500 MHz, CDCl3): d = 1.19 (t, 3H, J=7.5 Hz, SCH2CH3), 1.28 (s, 3H,
CH3), 1.35 (d, 3H, J5,6 =7.0 Hz, H-6), 1.49 (s, 3H, CH’3), 2.12 (s, 3H,
CH3C(O)CH2CH2C(O)O), 2.53–2.78 (m, 6H, CH3C(O)CH2CH2C(O)O,
SCH2CH3, CH3C(O)CH2CH2C(O)O), 3.78–3.82 (m, 1H, H-5), 3.98 (dd,
4.88 (d, 1H,
J1,2 =3.6 Hz, H-1), 5.16 (dd, 1H, J=1.2, 10.2 Hz,
OCH2CHCH2), 5.28 (dd, 1H, J=1.5, 17.1 Hz, OCH2CHCH’2), 5.87 (m,
1H, OCH2CHCH2); 13C NMR (75 MHz, CDCl3): d = 16.2 (C-6), [26.3,
28.3 (CH3CCH3)], 58.5 (OCH3), 63.1 (C-5), 68.2 (OCH2CHCH2), 75.7 (C-
4), 76.0 (C-3), 79.1 (C-2), 95.3 (C-1), 108.7 (CH3CCH3), 117.9
(OCH2CHCH2), 133.6 (OCH2CHCH2); MALDI-TOF/MS: m/z: calcd for
C13H22O5Na: 281.1365; found: 281.7 [M+Na]+.
Allyl 3-O-benzyl-2-O-methyl-a-d-fucopyranoside (7): Treatment of
(8.66 g, 33.53 mmol) in acetic acid/water (40.2 mL/26.8 mL) as described
in the general procedures gave the diol as white solid (7.39 g,
6
J
3,4 =5.5, J4,5 =2.5 Hz, H-4), 4.06 (dd, 1H, J2,3 =7.5, J3,4 =5.5 Hz, H-3),
a
4.25 (d, 1H, J1,2 =10.0 Hz, H-1), 4.92 (dd, 1H, J1,2 =10.0, J2,3 =7.5 Hz, H-
2); 13C NMR (75 MHz, CDCl3): d = 14.7 (SCH2CH3), 16.8 (C-6), [23.8,
26.4( CH3)], [27.8, 28.1 (CH3C(O)CH2CH2C(O)O, SCH2CH3)], 29.8
(CH3C(O)CH2CH2C(O)O), 38.0 (CH3C(O)CH2CH2C(O)O), 71.8 (C-2),
33.86 mmol, quantitative). Rf =0.30 (CH2Cl2/MeOH 19:1); [a]2D7 =+4.9
(c=2.5 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.21 (d, 3H, J5,6
6.6 Hz, H-6), 2.56 (s, 1H, OH), 3.40 (s, 3H, OCH3), 3.47 (dd, 1H, J1,2
=
=
3.0, J2,3 =9.6 Hz, H-2), 3.75 (s, 1H, H-4), 3.89–3.97 (m, 2H, H-3, H-5),
4.00 (dd, 1H, J=6.3, 12.6 Hz, OCH2CHCH2), 4.14 (dd, 1H, J=3.6,
12.9 Hz, OCH’2CHCH2), 4.99 (d, 1H, J1,2 =3.0 Hz, H-1), 5.16 (d, 1H, J=
10.5 Hz, OCH2CHCH2), 5.28 (d, 1H, J=17.1 Hz, OCH2CHCH’2), 5.87
(m, 1H, OCH2CHCH2); 13C NMR (75 MHz, CDCl3): d 16.1 (C-6), 57.7
(OCH3), 65.6 (C-5), 68.2 (OCH2CHCH2), 69.4(C-3), 71.5 (C-4), 77.9 (C-
2), 94.5 (C-1), 117.9 (OCH2CHCH2), 133.8 (OCH2CHCH2); MALDI-
TOF/MS: m/z: calcd for C10H18O5Na: 241.1052; found: 241.7 [M+Na]+.
72.7
(C-5),
76.4()C,-4 77.2
(C-3),
82.2
(C-1),
171.7
(CH3C(O)CH2CH2C(O)O), 206.3 (CH3C(O)CH2CH2C(O)O); MALDI-
TOF/MS: m/z: calcd for C16H26O6SNa: 369.13; found: 369.5 [M+Na]+.
Ethyl 3-O-benzyl-2-O-levulinoyl-1-thio-b-d-fucopyranoside (12): Treat-
ment of 11 (1.75 g, 5.05 mmol) in acetic acid/water (6.0 mL/4.0 mL) ac-
cording to the general procedure for isopropylidene removal gave the
diol as a white solid (1.55 g, quantitative). Rf =0.38(CH2Cl2/MeOH 19:1);
[a]2D7 =ꢀ3.5 (c=1.2 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.19 (t,
3H, J=7.5 Hz, SCH2CH3), 1.28 (d, 3H, J5,6 =6.0 Hz, H-6), 2.13 (s, 3H,
CH3C(O)CH2CH2C(O)O), 2.51–2.86 (m, 6H, CH3C(O)CH2CH2C(O)O,
SCH2CH3, CH3C(O)CH2CH2C(O)O), 3.57–3.68 (m, 2H, H-3, H-5), 3.75
(d, J=2.7 Hz, H-4), 4.32 (d, 1H, J1,2 =9.9 Hz, H-1), 4.98 (t, 1H, J1,2 =9.9,
Dibutyltin oxide (8.43 g, 33.86 mmol) was added to a solution of the diol
(7.39 g, 33.8 mmol) in dry MeOH (300 mL). The reaction mixture was
heated under reflux until the solution became clear. After cooling to
room temperature, the reaction mixture was concentrated to dryness.
J
2,3 =9.3 Hz, H-2); 13C NMR (75 MHz, CDCl3): d 14.8 (SCH2CH3), 16.6
Benzyl bromide (4.0 mL, 33.86 mmol) and CsF (5.15 g, 33.86 mmol) were
added to a solution of the residue in DMF (130 mL). The reaction mix-
ture was stirred at room temperature overnight, and then concentrated to
dryness. The residue was dissolved in CH2Cl2 (100 mL), and the solution
was washed with H2O (100 mL). The organic layer was dried (MgSO4),
filtered, and concentrated to dryness. Purification of the crude product
by column chromatography on silica gel (hexane/EtOAc 3:1) afforded
the desired product 7 as colorless oil (10.03 g, 32.53 mmol, 96%). Rf =
0.34(hexane/EtOAc 2:1); [ a]2D7 =+86.6 (c=2.0 in CHCl3); 1H NMR
(C-6), 23.7 (SCH2CH3), 28.2 (CH3C(O)CH2CH2C(O)O), 29.8
(CH3C(O)CH2CH2C(O)O), 38.4(CH 3C(O)CH2CH2C(O)O), [71.5, 71.8
(C-2,
(CH3C(O)CH2CH2C(O)O), 206.3 (CH3C(O)CH2CH2C(O)O); MALDI-
TOF/MS: m/z: calcd for C13H22O6SNa: 329.10; found: 330.2 [M+Na]+.
C-4)],
73.8
(C-3),
74.7
(C-5),
82.7
(C-1),
172.7
Dibutyl tin oxide (1.26 g, 5.06 mmol) was added to a solution of the diol
(1.55 g, 5.06 mmol) in dry toluene (50 mL). The reaction mixture was
heated under reflux with a Dean-Stark apparatus for 3 h, and then
cooled to 608C. Benzyl bromide (0.60 mL, 5.06 mmol) and tetrabutylam-
monium iodide (1.68 g, 5.06 mmol) were added and the resulting reaction
mixture was heated under reflux for 3 h. After cooling to room tempera-
ture, the reaction mixture was concentrated to dryness. The residue was
dissolved in EtOAc (50 mL), and the resulting solution was washed with
H2O (50 mL). The organic layer was dried (MgSO4) filtered, and concen-
trated to dryness. Purification of the crude product by column chroma-
tography on silica gel (hexane/EtOAc 2:1) afforded the desired product
(500 MHz, CDCl3): d
= 1.21 (d, 3H, J5,6 =6.5 Hz, H-6), 3.45 (s, 3H,
OCH3), 3.55 (dd, 1H, J1,2 =3.5, J2,3 =9.5 Hz, H-2), 3.59–3.78 (m, 2H, H-3,
H-5), 3.86 (dd, 1H, J2,3 =7.0, J3,4 =7.0 Hz, H-4), 3.99 (dd, 1H, J=7.0,
13.0 Hz, OCH2CHCH2), 4.12 (dd, 1H, J=5.5, 13.0 Hz, OCH’2CHCH2),
4.61 (d, 1H, J=12.0 Hz, PhCH2), 4.72 (d, 1H, J=12.0 Hz, PhCH’2), 4.94
(d, 1H, J1,2 =3.5 Hz, H-1), 5.15 (d, 1H, J=10.5 Hz, OCH2CHCH2), 5.26
(d, 1H, J=17.0 Hz, OCH2CHCH’2), 5.89 (m, 1H, OCH2CHCH2), 7.19–
7.29 (m, 5H, Harom); 13C NMR (75 MHz, CDCl3): d = 16.1 (C-6), 58.9
(OCH3), 65.3 (C-4), 68.2 (OCH2CHCH2), 70.2 (C-3), 72.7 (PhCH2), 77.5
(C-2), 77.9 (C-5), 95.5 (C-1), 117.9 (OCH2CHCH2), [127.7, 127.8, 128.4,
133.9 (Carom)], 138.3 (OCH2CHCH2); MALDI-TOF/MS: m/z: calcd for
C17H24O5Na: 331.1521; found: 331.2 [M+Na]+.
12 as colorless oil (1.04g, 52%). Rf =0.43 (hexane/EtOAc 1:1); [a]D27
=
ꢀ4.0 (c=0.8 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.16 (t, 3H,
J=7.5 Hz, SCH2CH3), 1.28 (d, 3H,
J5,6 =6.3 Hz, H-6), 2.12 (s, 3H,
CH3C(O)CH2CH2C(O)O), 2.48–2.76 (m, 6H, CH3C(O)CH2CH2C(O)O,
SCH2CH3, CH3C(O)CH2CH2C(O)O), 3.43–3.54 (m, 2H, H-3, H-5), 3.75
(d, J=3.0 Hz, H-4), 4.23 (d, 1H, J1,2 =9.9 Hz, H-1), 4.57 (d, 1H, J=
Allyl
4-azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-a-d-glucopyranoside
(8): Treatment of 7 (10.03 g, 32.53 mmol) in pyridine (28.6 mL, 0.33 mol)
and CH2Cl2 (160 mL) with trifluoromethanesulfonic anhydride (8.2 mL,
48.66 mmol) followed by treatment of triflate residue in DMF (400 mL)
with sodium azide (10.40 g, 0.16 mol) was performed according to the
general procedure to give compound 8 as colorless oil (8.67 g, 80%). Rf =
0.41 (hexane/EtOAc 5:1); [a]2D7 =+130.5 (c=2.4in CHCl 3); 1H NMR
12.0 Hz, PhCH2), 4.61 (d, 1H, J=11.1 Hz, PhCH’2), 5.14(t, 1H,
J1,2 =9.6,
J
2,3 =9.6 Hz, H-2), 7.19–7.31 (m, 5H, Harom); 13C NMR (75 MHz, CDCl3):
d
14.7
(SCH2CH3),
16.6
29.9
(C-6),
23.4(S
CH2CH3),
28.1
37.9
(CH3C(O)CH2CH2C(O)O),
(CH3C(O)CH2CH2C(O)O),
(CH3C(O)CH2CH2C(O)O), [69.1, 69.2 (C-2, C-4)], 71.7 (PhCH2), 74.5
(C-3), 79.7 (C-5), 82.9 (C-1), [127.9, 128.1, 128.5, 137.5 (Carom)], 171.7
(CH3C(O)CH2CH2C(O)O), 206.3 (CH3C(O)CH2CH2C(O)O); MALDI-
TOF/MS: m/z: calcd for C20H28O6SNa: 419.15; found: 419.5 [M+Na]+.
(500 MHz, CDCl3): d = 1.30 (d, 3H, J5,6 =6.5 Hz, H-6), 3.02 (t, 1H, J3,4
=
9.0, J4,5 =10.0 Hz, H-4), 3.27 (dd, 1H, J1,2 =3.5, J2,3 =9.5 Hz, H-2), 3.44 (s,
3H, OCH3), 3.52 (m, 1H, H-5), 3.72 (t, 1H, J2,3 =9.5, J3,4 =9.0 Hz, H-3),
3.98 (dd, 1H, J=7.0, 13.0 Hz, OCH2CHCH2), 4.12 (dd, 1H, J=5.0,
13.0 Hz, OCH’2CHCH2), 4.72 (d, 1H, J=10.5 Hz, PhCH2), 4.84 (d, 1H,
J=10.5 Hz, PhCH’2), 4.89 (d, 1H, J1,2 =3.5 Hz, H-1), 5.18 (d, 1H, J=
10.5 Hz, OCH2CHCH2), 5.28 (d, 1H, J=17.5 Hz, OCH2CHCH’2), 5.87
Ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-levulinoyl-1-thio-b-d-glucopyr-
anoside (13): Treatment of 12 (0.50 g, 1.26 mmol) in pyridine (1.0 mL,
12.61 mmol) and CH2Cl2 (6.5 mL) with trifluoromethanesulfonic anhy-
dride (0.32 mL, 1.90 mmol) followed by treatment of triflate residue in
Chem. Eur. J. 2006, 12, 9136 – 9149
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9143

G.-J. Boons, C. P. Quinn et al.
DMF (16 mL) with sodium azide (0.41 g, 6.31 mmol) according to the
general procedure for introduction of the C-4azide group gave com-
pound 13 as colorless oil (0.42 g, 79%). Rf =0.32 (hexane/EtOAc 4:1);
[a]2D7 =+13.1 (c=0.5 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.67
(t, 3H, J=7.5 Hz, SCH2CH3), 1.30 (d, 3H, J5,6 =5.7 Hz, H-6), 2.10 (s, 3H,
CH3C(O)CH2CH2C(O)O), 2.44–2.49 (m, 2H, CH3C(O)CH2CH2C(O)O),
2.57–2.67 (m, 4H, SCH2CH3, CH3C(O)CH2CH2C(O)O), 3.11–3.24(m,
oil (4.26 g, 78%). Rf =0.34(hexane/EtOAc 2:1); [ a]2D7 =+23.6 (c=1.8 in
CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.11 (d, 3H, J5,6 =6.0 Hz, H-
6b), 1.28 (d, 3H, J5,6 =6.0 Hz, H-6a), 1.72 (m, 2H, OCH2CH2CH2NHZ),
1.99 (s, 3H, CH3C(O)CH2CH2C(O)O), 2.32–2.39 (m, 2H,
CH3C(O)CH2CH2C(O)O), 2.50–2.67 (m, 2H, CH3C(O)CH2CH2C(O)O),
3.22 (dd, 2H, J=6.0, 12.3 Hz, OCH2CH2CH2NHZ), 3.39–3.49 (m, 2H,
J
3,4 =9.6, J4,5 =9.6 Hz, OCH2CH2CH2NHZ, H-4b), 3.56 (t, J3,4 =9.3, J4,5 =
9.3 Hz, H-4a), 3.62–3.72 (m, 2H, OCH’2CH2CH2NHZ, H-5a), 3.84(m,
1H, H-5b), 4.17 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.45 (d, 1H, J=
11.4Hz, PhC H2), 4.50 (d, 1H, J=11.4Hz, PhC H’2), 4.66 (d, 1H, J=
10.8 Hz, PhCH’’2), 4.80 (broad, 2H, H-1a, NH), 4.95 (d, 1H, J=10.8 Hz,
PhCH’’’2), 4.99 (s, 2H, PhCH2OC(O)), 5.06 (s, 1H, H-1b), 5.29 (d, 1H,
2H, H-4, H-5), 3.48 (t, J3,4 =9.0, J2,3 =9.0 Hz, H-3), 4.27 (d, 1H, J1,2
=
9.9 Hz, H-1), 4.68 (d, 1H, J=11.1 Hz, PhCH2), 4.71 (d, 1H, J=11.1 Hz,
PhCH’2), 4.94 (dd, 1H, J1,2 =9.9, J2,3 =9.0 Hz, H-2), 7.22–7.29 (m, 5H,
H
arom); 13C NMR (75 MHz, CDCl3): d 14.8 (SCH2CH3), 18.7 (C-6), 23.9
(SCH2CH3),
28.0
(CH3C(O)CH2CH2C(O)O),
29.8
J
2,3 =3.3 Hz, H-2a), 5.32 (dd, 1H, J2,3 =3.0, J3,4 =9.6 Hz, H-3b), 5.52 (dd,
1H, J1,2 =1.8, J2,3 =3.0 Hz, H-2b), 7.05–8.00 (m, 25H, Harom); 13C NMR
(75 MHz, CDCl3): 17.8 (C-6b), 18.2 (C-6a), 28.0
29.7 (OCH2CH2CH2NHZ,
(CH3C(O)CH2CH2C(O)O), 38.5
(CH3C(O)CH2CH2C(O)O), 37.8
72.2 (C-2), 74.9 (PhCH2), 75.1 (C-5), 82.3 (C-3), 83.2 (C-1), [127.9, 128.0,
128.2, 128.4, 137.5 (Carom)], 171.5 (CH3C(O)CH2CH2C(O)O), 206.1
AHCTREUNG
d
=
(CH3C(O)CH2CH2C(O)O),
CH3C(O)CH2CH2C(O)O)),
(CH3C(O)CH2CH2C(O)O);
MALDI-TOF/MS:
m/z:
calcd
for
C20H27N3O5SNa: 444.15; found: 444.1 [M+Na]+.
37.8
ACHTREUNG
(OCH2CH2CH2NHZ), 65.6 (OCH2CH2CH2NHZ), 66.5 (PhCH2OC(O)),
67.9 (C-5a), 68.6 (C-5b), 70.7 (C-2b), 71.8 (C-3b), 72.7 (C-2a), [73.9, 75.8
(PhCH2)], 78.2 (C-4b), 79.3 (C-3a), 79.8 (C-4a), 97.0 (C-1a), 99.7 (C-1b),
[127.5, 127.7, 127.9, 128.2, 128.3, 128.4, 128.5, 129.5, 129.6, 129.7, 129.8,
133.4, 136.6, 137.9, 138.0 (Carom)], 156.3 (PhCH2OC(O)), [165.3, 166.1
Ethyl 2-O-benzoyl-4-O-benzyl-3-O-levulinoyl-1-thio-a-l-rhamnopyrano-
side (15): Treatment of 14 (4.93 g, 12.25 mmol) and levulinic acid
(12.5 mL, 122.50 mmol) in CH2Cl2 (180 mL) with DCC (15.18 g,
73.57 mmol) and DMAP (22.45 mg, 0.18 mmol) in CH2Cl2 (18 mL) ac-
cording to the general procedure for levulination gave compound 15 as
colorless oil (5.29 g, 86%). Rf =0.34(hexane/EtOAc 3:1); [ a]2D7 =ꢀ18.9
(PhC(O)O)],
171.7
(CH3C(O)CH2CH2C(O)O),
206.2
(CH3C(O)CH2CH2C(O)O); MALDI-TOF/MS: m/z: found: 1011.6
[M+Na]+; MALDI-FTICR/MS: m/z: calcd for C56H61NO15Na:
1010.3939; found: 1010.3932 [M+Na]+.
1
(c=2.6 in CHCl3); H NMR (300 MHz, CDCl3): d 1.23 (t, 3H, J=7.2 Hz,
SCH2CH3), 1.33 (d, 3H,
CH3C(O)CH2CH2C(O)O), 2.35–2.39 (td, 2H, J=6.9, 9.6 Hz,
CH3C(O)CH2CH2C(O)O), 2.46–2.72 (m, 4H, SCH2CH3,
J5,6 =6.0 Hz, H-6), 2.02 (s, 3H,
3-[(N-Benzyloxycarbonyl)amino]propyl O-(2-O-benzoyl-4-O-benzyl-a-l-
rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-rhamnopyranoside
(18): Treatment of 17 (4.26 g, 4.31 mmol) in CH2Cl2 (100 mL) with hydra-
zine acetate (397 mg, 4.31 mmol) in MeOH (10 mL) according to the
general procedure for cleavage of the levulinoyl ester gave compound 18
as white solid (3.56 g, 93%). Rf =0.42 (hexane/EtOAc 2:1); [a]2D7 =+21.9
CH3C(O)CH2CH2C(O)O), 3.58 (t, J4,5 =9.3, J3,4 =9.6 Hz, H-4), 4.18 (m,
1H, H-5), 4.59 (d, 1H, J=11.1 Hz, PhCH2), 4.66 (d, 1H, J=11.1 Hz,
PhCH’2), 5.22 (s, 1H, H-1), 5.28 (dd, 1H, J2,3 =3.3, J3,4 =9.6 Hz, H-3), 5.51
(dd, 1H, J1,2 =1.5, J2,3 =3.3 Hz, H-2), 7.19–8.00 (m, 10H, Harom); 13C NMR
(75 MHz, CDCl3): d 14.9 (SCH2CH3), 18.0 (C-6), 25.4(S CH2CH3), 27.9
(CH3C(O)CH2CH2C(O)O), 29.7 (CH3C(O)CH2CH2C(O)O), 37.8-
(c=2.2 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 1.17 (d, 3H, J5,6
6.0 Hz, H-6b), 1.26 (d, 3H, 5,6 =6.0 Hz, H-6a), 1.72 (m, 2H,
=
J
(CH3C(O)CH2CH2C(O)O), 68.3 (C-5), 72.5 (C-2), 72.6 (C-3), 74.9
OCH2CH2CH2NHZ), 3.22 (d, 2H, J=6.0 Hz, OCH2CH2CH2NHZ), 3.30–
3.41 (m, 2H, J3,4 =9.6, J4,5 =9.3 Hz, OCH2CH2CH2NHZ, H-4b), 3.54 (t,
1H, J3,4 =9.3, J4,5 =9.3 Hz, H-4a), 3.62–3.71 (m, 2H, OCH’2CH2CH2NHZ,
(PhCH2), 78.9 (C-4), 81.9 (C-1), [127.8, 127.9, 128.4, 128.5, 129.7, 129.8,
133.4, 137.9 (Carom)], 165.5 (PhC(O)O), 171.7 (CH3C(O)CH2CH2C(O)O),
206.2 (CH3C(O)CH2CH2C(O)O); MALDI-TOF/MS: m/z: found: 524.1
[M+Na]+; MALDI-FTICR/MS: m/z: calcd for C27H32O7SNa: 523.1766;
found: 523.1761 [M+Na]+.
H-5a), 3.78 (dd, 1H, J4,5 =9.3 Hz, J5,6 =6.0 Hz, H-5b), 4.04 (dd, 1H, J2,3
=
2.1, J3,4 =9.6 Hz, H-3b), 4.18 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.57–
4.63 (m, 3H, PhCH2), 4.77 (s, 1H, H-1a), 4.86 (d, 1H, J=10.8 Hz,
PhCH’2), 4.99 (s, 2H, PhCH2OC(O)), 5.11 (s, 1H, H-1b), 5.28 (d, 1H,
3-[(N-Benzyloxycarbonyl)amino]propyl
2-O-benzoyl-4-O-benzyl-a-l-
rhamnopyranoside (16): Glycosyl donor 14 (3.79 g, 9.42 mmol), 3-(N-ben-
zyloxycarbonyl)aminopropanol (3.94g, 18.83 mmol) and 4ꢃ powdered
molecular sieves (7.73 g) in CH2Cl2 (150 mL) in the presence of NIS
(2.33 g, 10.36 mmol) and TfOH (0.166 mL, 1.88 mmol) were reacted ac-
cording to the general procedure for NIS glycosylation to give compound
J
2,3 =3.0 Hz, H-2a), 5.33 (dd, 1H, J2,3 =2.1 Hz, H-2b), 7.12–8.01 (m, 25H,
Harom); 13C NMR (75 MHz, CDCl3): d 17.9 (C-6b), 18.1 (C-6a), 29.5
(OCH2CH2CH2NHZ), 38.5 (OCH2CH2CH2NHZ), 65.6
(OCH2CH2CH2NHZ), 66.5 (PhCH2OC(O)), 67.9 (C-5a), 68.3 (C-5b),
69.8 (C-3b), 72.8 (C-2b), 73.1 (C-2a), [74.0, 75.6 (PhCH2)], 77.6 (C-3a),
80.3 (C-4a), 81.1 (C-4b), 97.1 (C-1a), 99.5 (C-1b), [127.7, 127.8, 127.9,
128.2, 128.3, 128.4, 128.5, 129.6, 129.7, 129.8, 133.2, 133.3, 137.8, 138.1
(Carom)], 156.3 (PhCH2OC(O)), [165.8, 165.9 (PhC(O)O)]; MALDI-TOF/
MS: m/z: found: 913.5 [M+Na]+; MALDI-FTICR/MS: m/z: calcd for
C51H55NO13Na: 912.3571; found: 912.3559 [M+Na]+.
16 as white solid (3.73 g, 72%). Rf =0.26 (hexane/EtOAc 2:1); [a]2D7 =+
1
11.3 (c=1.8 in CHCl3); H NMR (300 MHz, CDCl3): d 1.32 (d, 3H, J5,6
=
6.0 Hz, H-6), 1.73 (m, 2H, OCH2CH2CH2NHZ), 2.11 (d, 1H, J=4.5 Hz,
OH), 3.24(dd, 2H, J=6.3, 12.6 Hz, OCH2CH2CH2NHZ), 3.36–3.45 (m,
2H, OCH2CH2CH2NHZ, H-4), 3.65–3.75 (m, 2H, OCH’2CH2CH2NHZ,
H-5), 4.12 (dd, 1H, J2,3 =3.3, J3,4 =8.4Hz, H-3), 4.69 (d, 1H, J=11.1 Hz,
PhCH2), 4.76 (s, 1H, H-1), 4.79 (d, 1H, J=11.1 Hz, PhCH’2), 4.85 (broad,
3-[(N-Benzyloxycarbonyl)amino]propyl
O-(4-azido-3-O-benzyl-4,6-di-
deoxy-2-O-levulinoyl-b-d-glucopyranosyl)-(1!3)-O-(2-O-benzoyl-4-O-
benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-rham-
nopyranoside (19): Glycosyl donor 13 (80 mg, 0.19 mmol), glycosyl ac-
ceptor 18 (151 mg, 0.17 mmol) and 4ꢃ powdered molecular sieves
(0.23 g) in CH2Cl2 (3 mL) in the presence of NIS (47 mg, 0.21 mmol) and
TfOH (3 mL, 0.034mmol) was treated according to the general procedure
for the linker glycosylation to give compound 19 as colorless oil (161 mg,
76%). Rf =0.30 (hexane/EtOAc 2:1); [a]2D7 =+15.3 (c=0.8 in CHCl3);
1H NMR (500 MHz, CDCl3): d = 0.89 (d, 3H, J5,6 =6.5 Hz, H-6c), 1.06
(d, 3H, J5,6 =6.5 Hz, H-6b), 1.27 (d, 3H, J5,6 =6.5 Hz, H-6a), 1.73 (m, 2H,
OCH2CH2CH2NHZ), 1.88 (s, 3H, CH3C(O)CH2CH2C(O)O), 1.99–2.10
1H, NH), 5.02 (s, 2H, PhCH2OC(O)), 5.25 (dd, 1H, J1,2 =1.5, J2,3
=
3.3 Hz, H-2), 7.18–7.99 (m, 15H, Harom); 13C NMR (75 MHz, CDCl3): d
18.2 (C-6), 29.6 (OCH2CH2CH2NHZ), 38.6 (OCH2CH2CH2NHZ), 65.6
(OCH2CH2CH2NHZ), 66.6 (C-5), 67.6 (PhCH2OC(O)), 70.5 (C-3), 73.2
(C-2), 75.2 (PhCH2), 81.6 (C-4), 97.5 (C-1), [127.9, 128.1, 128.4, 129.7,
129.9, 130.4, 133.3, 136.6, 138.1 (Carom)], 156.3 (PhCH2OC(O)), 166.3-
(PhC(O)O); MALDI-TOF/MS: m/z: found: 572.9 [M+Na]+; MALDI-
FTICR/MS: m/z: calcd for C31H35NO8Na: 572.2260; found: 572.2259
[M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl O-(2-O-benzoyl-4-O-benzyl-3-O-
levulinoyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-
rhamnopyranoside (17): Glycosyl donor 15 (3.04g, 6.07 mmol), glycosyl
acceptor 16 (3.03 g, 5.51 mmol) and 4ꢃ powdered molecular sieves
(6.07 g) in CH2Cl2 (100 mL) in the presence of NIS (1.51 g, 6.71 mmol)
and TfOH (0.11 mL, 1.22 mmol) was treated according to the general
procedure for the linker glycosylation to give compound 17 as colorless
(m,
CH3C(O)CH2CH2C(O)O), 2.76 (m, 1H, H-5c), 2.91 (t, 1H, J3,4 =9.5,
4,5 =10.0 Hz, H-4c), 3.17–3.23 (m, 3H, OCH2CH2CH2NHZ, H-3c), 3.41–
3.44 (m, 2H, OCH2CH2CH2NHZ, H-4a), 3.56 (t, 1H, J3,4 =9.5, J4,5
2H,
CH3C(O)CH2CH2C(O)O),
2.12–2.22
(m,
2H,
J
=
9.5 Hz, H-4b), 3.66–3.74 (m, 3H, OCH’2CH2CH2NHZ, H-5a, H-5b), 3.97
(dd, 1H, J2,3 =3.0, J3,4 =9.5 Hz, H-3a), 4.20 (m, 2H, H-1c, H-3b), 4.45 (d,
9144
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9136 – 9149

Anthrax Oligosaccharides
FULL PAPER
1H, J=12.0 Hz, PhCH2), 4.53 (d, 1H, J=11.5 Hz, PhCH’2), 4.61 (d, 1H,
J=12.0 Hz, PhCH’’2), 4.63 (d, 1H, J=11.5 Hz, PhCH’’’2), 4.71 (d, 1H, J=
11.5 Hz, PhCH’’’’2), 4.77 (s, 1H, H-1a), 4.84 (m, 2H, NH, PhCH’’’’’2), 4.95
(t, 1H, J1,2 =8.0, J2,3 =10.5 Hz, H-2c), 5.00 (s, 2H, PhCH2OC(O)), 5.14(s,
1H, H-1b), 5.30 (d, 1H, J2,3 =3.0 Hz, H-2a), 5.32 (d, 1H, J2,3 =3.0 Hz, H-
2b), 7.13–8.05 (m, 30H, Harom); 13C NMR (75 MHz, CDCl3): d 17.7 (C-
6c), 17.9 (C-6b), 18.1 (C-6a), 27.6 (CH3C(O)CH2CH2C(O)O), 29.6
1.37 mmol), donor 9 (0.622 g, 1.51 mmol) and 4ꢃ powdered molecular
sieves (1.85 g) in dry acetonitrile (23 mL) was stirred at 08C for 1 h, and
then cooled to ꢀ408C. A solution of BF3·Et2O (0.28 mL, 2.27 mmol) was
added slowly. The mixture was stirred at ꢀ408C for 1 h, and then neutral-
ized with triethylamine. The solution was filtered through Celite, washed
with MeOH/CH2Cl2 5:95 (20 mL), and the combined filtrates were con-
centrated to dryness. Purification of the crude product by column chro-
matography (hexane/EtOAc 3:1) on silica gel afforded the desired prod-
uct 21 as a/b 1:4mixture (1.38 g, 86%).
(CH3C(O)CH2CH2C(O)O),
(CH3C(O)CH2CH2C(O)O),
31.6
38.5
(OCH2CH2CH2NHZ),
(OCH2CH2CH2NHZ),
37.3
65.6
(OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 67.2 (C-4c), 67.8 (C-5a), 68.6
(C-5b), 70.6 (C-5c), 71.9 (C-2a), 72.7 (C-2b), [73.4, 74.3, 74.4 (PhCH2)],
75.4 (C-2c), 77.2 (C-3b), 78.0 (C-3a), 79.6 (C-4a), 80.2 (C-4b), 80.9 (C-
3c), 97.2 (C-1a), 98.8 (C-1b), 100.3 (C-1c), [127.0, 127.3, 127.8, 128.0,
128.1, 128.2, 128.3, 128.4, 128.5, 129.8, 129.9, 130.1, 133.0, 133.3, 133.4,
136.6, 137.5, 137.9, 138.6 (Carom)], 156.3 (PhCH2OC(O)), [165.7, 165.8
Method B: Methyl iodide (0.20 mL, 3.24mmol) and silver( i) oxide
(0.37 g, 1.60 mmol) were added to a solution of 20 (93 mg, 0.08 mmol) in
THF (2 mL). Dimethyl sulfide (1 mL, 0.014mmol) was added as catalyst.
The flask was wrapped by aluminium foil to exclude light. The reaction
mixture was stirred at room temperature overnight, and then filtered
through Celite. The filtrate was concentrated to dryness. Purification of
the crude product by column chromatography (hexane/EtOAc 3:1) on
silica gel afforded the desired product 21 as colorless oil (48 mg, 51%).
Rf =0.56 (hexane/EtOAc 2:1); [a]2D7 =+82.0 (c=0.2 in CHCl3); 1H NMR
(500 MHz, CDCl3): d = 0.83 (d, 3H, J5,6 =6.0 Hz, H-6c), 1.12 (d, 3H,
(PhC(O)O)],
171.1
(CH3C(O)CH2CH2C(O)O),
206.1
(CH3C(O)CH2CH2C(O)O); MALDI-TOF/MS: m/z: found: 1271.7
[M+Na]+; MALDI-FTICR/MS: m/z: calcd for C69H76N4O18Na:
1271.5052; found: 1271.4893 [M+Na]+.
J
5,6 =6.0 Hz, H-6b), 1.24(d, 3H,
OCH2CH2CH2NHZ), 2.74(m, 1H, H-5c), 2.84(t, 1H,
10.0 Hz, H-4c), 2.91 (t, 1H, J1,2 =8.0, J2,3 =9.0 Hz, H-2c), 3.11 (t, 1H,
2,3 =9.0, J3,4 =9.5 Hz, H-3c), 3.22 (m, 2H, OCH2CH2CH2NHZ), 3.36 (s,
3H, OCH3), 3.39 (m, 1H, OCH2CH2CH2NHZ), 3.50 (t, 1H, J3,4 =9.5,
4,5 =10.0 Hz, H-4b), 3.54 (t, 1H, J3,4 =9.5, J4,5 =10.0 Hz, H-4a), 3.67 (m,
2H, OCH’2CH2CH2NHZ, H-5a), 3.76 (m, 1H, H-5b), 4.10 (dd, 1H, J2,3
J
5,6 =5.5 Hz, H-6a), 1.73 (m, 2H,
3-[(N-Benzyloxycarbonyl)amino]propyl
O-(4-azido-3-O-benzyl-4,6-di-
J
3,4 =10.5, J4,5
=
deoxy-b-d-glucopyranosyl)-(1!3)-O-(2-O-benzoyl-4-O-benzyl-a-l-rham-
nopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-rhamnopyranoside
(20): Treatment of 19 (116 mg, 0.093 mmol) in CH2Cl2 (2.3 mL) with hy-
drazine acetate (8.6 mg, 0.093 mmol) in MeOH (0.23 mL) according to
the general procedure for cleavage of the levulinoyl ester gave compound
20 as white solid (100 mg, 93%). Rf =0.36 (hexane/EtOAc 2:1); [a]2D7 =+
11.0 (c=0.06 in CHCl3); 1H NMR (500 MHz, CDCl3): d = 0.88 (d, 3H,
J
J
=
3.0, J3,4 =9.5 Hz, H-3b), 4.20 (dd, 1H, J2,3 =3.0, J3,4 =9.5 Hz, H-3a), 4.31
(d, 1H, J1,2 =8.0 Hz, H-1c), 4.52 (d, 1H, J=11.0 Hz, PhCH2), 4.59 (d, 1H,
J=10.5 Hz, PhCH’2), 4.65 (d, 1H, J=11.0 Hz, PhCH’’2), 4.72 (d, 1H, J=
11.5 Hz, PhCH’’’2), 4.75 (d, 1H, J=12.0 Hz, PhCH’’’’2), 4.76 (s, 1H, H-
1a), 4.80 (d, 1H, J=10.5 Hz, PhCH’’’’’2), 4.82 (broad, 1H, NH), 5.00 (s,
2H, PhCH2OC(O)), 5.14(s, 1H, H-1b), 5.30 (d, 1H,
5.38 (d, 1H, J2,3 =3.0 Hz, H-2b), 7.19–8.02 (m, 30H, Harom); 13C NMR
(75 MHz, CDCl3): 17.8 (C-6c), 18.0 (C-6b), 18.1 (C-6a), 29.6
(OCH2CH2CH2NHZ), 38.5 (OCH2CH2CH2NHZ), 60.4(OCH 3), 65.6
(OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 67.3 (C-5c), 67.9 (C-5a), 68.6
(C-5b), 70.2 (C-4c), 72.7 (C-2a), 73.2 (C-2b), [74.2, 75.2, 75.5 (PhCH2)],
75.9 (C-3b), 78.0 (C-3a), 80.0 (C-4a), 80.5 (C-4b), 82.5 (C-3c), 84.3 (C-
2c), 97.1 (C-1a), 99.2 (C-1b), 102.9 (C-1c), [127.6, 127.8, 127.9, 128.0,
128.2, 128.3, 128.4, 128.5, 129.7, 129.8, 129.9, 130.1, 133.0, 133.3, 137.8,
137.9, 138.2 (Carom)], 156.3 (PhCH2OC(O)), [165.5, 165.8 (PhC(O)O)];
MALDI-TOF/MS: m/z: found: 1187.8 [M+Na]+; MALDI-FTICR/MS:
m/z: calcd for C65H72N4O16Na: 1187.4841; found: 1187.4715 [M+Na]+.
J
5,6 =6.0 Hz, H-6c), 1.13 (d, 3H, J5,6 =6.0 Hz, H-6b), 1.26 (d, 3H, J5,6
=
5.5 Hz, H-6a), 1.73 (m, 2H, OCH2CH2CH2NHZ), 2.74(m, 1H, H-5c),
2.83 (t, 1H, J3,4 =9.5, J4,5 =10.0 Hz, H-4c), 3.12 (t, 1H, J2,3 =9.0, J3,4
9.5 Hz, H-3c), 3.22 (m, 2H, OCH2CH2CH2NHZ), 3.30 (t, 1H, J1,2 =8.0,
=
J2,3 =3.0 Hz, H-2a),
J
2,3 =9.0 Hz, H-2c), 3.40 (m, 1H, OCH2CH2CH2NHZ), 3.49 (t, 1H, J3,4 =
9.0, J4,5 =10.0 Hz, H-4b), 3.54 (t, 1H, J3,4 =9.0, J4,5 =10.0 Hz, H-4a), 3.64–
3.71 (m, 2H, OCH’2CH2CH2NHZ, H-5a), 3.78 (m, 1H, H-5b), 4.05 (dd,
1H, J2,3 =3.0, J3,4 =9.5 Hz, H-3b), 4.08 (d, 1H, J1,2 =8.0 Hz, H-1c), 4.21
(dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.56 (d, 1H, J=10.5 Hz, PhCH2),
4.60 (d, 1H, J=10.5 Hz, PhCH’2), 4.66 (d, 1H, J=11.0 Hz, PhCH’’2), 4.71
(d, 1H, J=12.0 Hz, PhCH’’’2), 4.75 (d, 1H, J=12.0 Hz, PhCH’’’’2), 4.76 (s,
1H, H-1a), 4.83 (broad, 1H, NH), 4.92 (d, 1H, J=11.0 Hz, PhCH’’’’’2),
5.00 (s, 2H, PhCH2OC(O)), 5.12 (s, 1H, H-1b), 5.32 (d, 1H, J2,3 =3.0 Hz,
d
H-2a), 5.36 (d, 1H,
13C NMR (75 MHz, CDCl3):
(OCH2CH2CH2NHZ), 38.5
J
2,3 =3.0 Hz, H-2b), 7.19–8.01 (m, 30H, Harom);
17.9 (C-6c), 18.1 (C-6a, C-6b), 29.6
(OCH2CH2CH2NHZ), 65.6
d
3-[(N-Benzyloxycarbonyl)amino]propyl O-(4-(3-hydroxy-3-methylbuta-
namido)-3-O-benzyl-4,6-dideoxy-2-O-methyl-b-d-glucopyranosyl)-(1!3)-
O-(2-O-benzoyl-4-O-benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-
O-benzyl-a-l-rhamnopyranoside (22): Treatment of 21 (0.71 g,
0.61 mmol), 1,3-propanedithiol (1.26 mL, 12.55 mmol) in pyridine
(43 mL) and H2O (6.1 mL) with TEA (1.28 mL, 9.15 mmol) according to
the general procedure for azide reduction and introduction of C-4’’ moit-
ety gave free amine(0.69 g, 99%). Treatment of the free amine (0.47 g,
0.41 mmol) in DMF (20 mL) with b-hydroxyisovaleric acid (88 mL,
0.82 mmol) which was activated with HOAt (0.23 g, 1.64mmol) and
HATU (0.62 g, 1.64mmol) in DMF (10 mL) for 1 h, and then added
DIPEA (5.71 mL, 3.28 mmol) gave compound 22 as colorless oil (0.32 g,
63%) and its a-isomer (76 mg, 15%). Rf =0.26 (hexane/EtOAc 1:1);
(OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 66.9 (C-5c), 67.9 (C-5a), 68.7
(C-5b), 70.6 (C-4c), 72.5 (C-2a, C-2b), 74.6 (C-2c), [74.7, 75.0, 75.3
(PhCH2)], 75.4 (C-3b), 77.6 (C-3a), 80.1 (C-4a, C-4b), 82.2 (C-3c), 97.1
(C-1a), 99.0 (C-1b), 103.0 (C-1c), [127.8, 127.9, 128.0, 128.1, 128.2, 128.3,
128.4, 128.5, 128.6, 129.8, 130.0, 133.3, 133.4, 136.6, 137.8, 138.0 (Carom)],
156.4(PhCH 2OC(O)), [165.6, 165.8 (PhC(O)O)]; MALDI-TOF/MS: m/
z: found: 1172.7 [M+Na]+; MALDI-FTICR/MS: m/z: calcd for
C64H70N4O16Na: 1173.4685; found: 1173.4588 [M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl
O-(4-azido-3-O-benzyl-4,6-di-
deoxy-2-O-methyl-b-d-glucopyranosyl)-(1!3)-O-(2-O-benzoyl-4-O-
benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-rham-
nopyranoside (21)
1
[a]2D7 =+9.4( c=0.3 in CHCl3); H NMR (500 MHz, CDCl3): d = 0.73 (d,
Method A: Sodium acetate (0.63 g, 7.68 mmol) and PdCl2 (0.38 g,
2.14mmol) was added to a solution of 8 (0.594g, 1.78 mmol) in AcOH/
H2O 9:1 (60 mL). The reaction mixture mixture was stirred at room tem-
perature overnight, and then filtered through Celite. The filtrate was con-
centrated to dryness and the residue was co-evaporated with toluene (2ꢂ
60 mL). Purification of the crude product by column chromatography on
silica gel (hexane/EtOAc 2:1) afforded the hemiacetal compound. To a
solution of this compound in CH2Cl2 (30 mL) was added trichloroacetoni-
trile (1.79 mL, 17.85 mmol) and DBU (0.11 mL, 0.74mmol). The reaction
mixture was stirred at room temperature for 5 h, and then concentrated
to dryness. Purification of the crude product by column chromatography
on silica gel (hexane/EtOAc 2:1 +0.5%TEA) afforded imidate donor 9
as an a/b mixture 9:1 (0.622 g, 85%). A mixture of acceptor 18 (1.22 g,
3H, J5,6 =5.5 Hz, H-6c), 1.09 (s, 3H, (CH3)2C(OH)CH2C(O)NH), 1.12 (d,
3H, J5,6 =6.0 Hz, H-6b), 1.18 (s, 3H, (CH’3)2C(OH)CH2C(O)NH), 1.24
(d, 3H, J5,6 =5.5 Hz, H-6a), 1.74(m, 2H, OCH 2CH2CH2NHZ), 1.99 (d,
1H, J=15.0 Hz, (CH3)2C(OH)CH2C(O)NH), 2.09 (d, 1H, J=15.0 Hz,
(CH3)2C(OH)CH’2C(O)NH), 2.91 (m, 1H, H-5c), 2.98 (t, 1H, J1,2 =8.0,
J
2,3 =8.5 Hz, H-2c), 3.15 (t, 1H, J2,3 =8.5, J3,4 =9.0 Hz, H-3c), 3.22 (m, 2H,
OCH2CH2CH2NHZ), 3.38 (s, 3H, OCH3), 3.39 (m, 2H,
OCH2CH2CH2NHZ, H-4c), 3.52 (t, 1H, J3,4 =9.0, J4,5 =9.5 Hz, H-4b), 3.54
(t, 1H, J3,4 =9.0, J4,5 =9.5 Hz, H-4a), 3.67 (m, 2H, OCH’2CH2CH2NHZ,
H-5a), 3.76 (m, 1H, H-5b), 4.12 (dd, 1H, J2,3 =3.5, J3,4 =9.0 Hz, H-3b),
4.21 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.34 (d, 1H, J1,2 =8.0 Hz, H-
1c), 4.48 (d, 1H, J=11.0 Hz, PhCH2), 4.54 (d, 1H, J=11.0 Hz, PhCH’2),
Chem. Eur. J. 2006, 12, 9136 – 9149
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9145

G.-J. Boons, C. P. Quinn et al.
4.60 (d, 1H, J=10.5 Hz, PhCH’’2), 4.71 (d, 1H, J=12.5 Hz, PhCH’’’2),
4.77 (s, 1H, H-1a), 4.83 (d, 1H, J=11.5 Hz, PhCH’’’’2), 4.85 (broad, 1H,
NH), 4.95 (d, 1H, J=11.0 Hz, PhCH’’’’’2), 5.00 (s, 2H, PhCH2OC(O)),
+18.3 (c=0.6 in CHCl3); 1H NMR (300 MHz, CDCl3): d = 0.73 (d, 3H,
J
5,6 =6.0 Hz, H-6c), 0.78 (s, 3H, (CH3)2CHCH2C(O)NH), 0.82 (s, 3H,
(CH’3)2CHCH2C(O)NH), 1.12 (d, 3H, J5,6 =6.0 Hz, H-6b), 1.24(d, 3H,
5,6 =5.5 Hz, H-6a), 1.71 (m, 2H, OCH2CH2CH2NHZ), 1.80–1.98 (m, 3H,
(CH3)2CHCH2C(O)NH, (CH3)2CHCH2C(O)NH), 2.92 (m, 1H, H-5c),
2.97 (t, 1H, 1,2 =7.8, 2,3 =9.0 Hz, H-2c), 3.15–3.23 (m, 3H,
5.15 (s, 1H, H-1b), 5.30 (d, 1H, J2,3 =3.0 Hz, H-2a), 5.39 (d, 1H, J2,3
=
J
3.5 Hz, H-2b), 7.14–8.00 (m, 30H, Harom); 13C NMR (75 MHz, CDCl3): d
= 17.7 (C-6c), 17.8 (C-6b), 18.1 (C-6a), 29.2 (OCH2CH2CH2NHZ), [29.3,
29.7 ((CH3)2C(OH)CH2C(O)NH)], 38.5 (OCH2CH2CH2NHZ), 47.7
((CH3)2C(OH)CH2C(O)NH), 55.7 (C-4c), 60.3 (OCH3), 65.6
(OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 67.9 (C-5a), 68.6 (C-5b),
69.4((CH 3)2C(OH)CH2C(O)NH), 70.6 (C-5c), 72.8 (C-2a), 73.1 (C-2b),
[73.5, 74.1, 75.5 (PhCH2)], 76.1 (C-3b), 78.1 (C-3a), 79.8 (C-3c), 80.0 (C-
4a), 80.5 (C-4b), 84.4 (C-2c), 97.1 (C-1a), 99.2 (C-1b), 103.0 (C-1c),
[127.5, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 129.7,
129.8, 129.9, 130.2, 133.0, 133.3, 137.9, 138.3, 138.5 (Carom)], 156.4
J
J
OCH2CH2CH2NHZ, H-3c), 3.37 (s, 3H, OCH3), 3.39 (m, 2H,
OCH2CH2CH2NHZ, H-4c), 3.51 (t, 1H, J3,4 =9.0, J4,5 =9.0 Hz, H-4b), 3.56
(t, 1H, J3,4 =9.0, J4,5 =9.0 Hz, H-4a), 3.66 (m, 2H, OCH’2CH2CH2NHZ,
H-5a), 3.76 (m, 1H, H-5b), 4.13 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3b),
4.21 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.34 (d, 1H, J1,2 =7.8 Hz, H-
1c), 4.48 (d, 1H, J=12.0 Hz, PhCH2), 4.53 (d, 1H, J=10.8 Hz, PhCH’2),
4.60 (d, 1H, J=10.8 Hz, PhCH’’2), 4.69 (d, 1H, J=12.0 Hz, PhCH’’’2),
4.77 (s, 1H, H-1a), 4.83 (d, 1H, J=10.8 Hz, PhCH’’’’2), 4.85 (broad, 1H,
NH), 4.96 (d, 1H, J=10.8 Hz, PhCH’’’’’2), 5.00 (s, 2H, PhCH2OC(O)),
5.15 (d, 1H, J1,2 =1.2 Hz, H-1b), 5.30 (dd, 1H, J1,2 =1.2, J2,3 =3.0 Hz, H-
(PhCH2OC(O)),
[165.6,
165.9
(PhC(O)O)],
172.2
((CH3)2C(OH)CH2C(O)NH); MALDI-TOF/MS: m/z: found: 1261.4;
MALDI-FTICR/MS: m/z: calcd for C70H82N2O18Na: 1261.5460; found:
1261.5427 [M+Na]+.
2a), 5.39 (dd, 1H,
J1,2 =1.8, J2,3 =3.0 Hz, H-2b), 7.18–8.06 (m, 30H,
Harom); 13C NMR (75 MHz, CDCl3): d = 17.6 (C-6c), 17.8 (C-6b), 18.1
(C-6a),
((CH3)2CHCH2C(O)NH),
[22.4,
22.5
((CH3)2CHCH2C(O)NH)],
(OCH2CH2CH2NHZ),
25.9
38.4
3-[(N-Benzyloxycarbonyl)amino]propyl O-(4-(3-hydroxy-3-methylbuta-
namido)-3-O-benzyl-4,6-dideoxy-b-d-glucopyranosyl)-(1!3)-O-(2-O-ben-
zoyl-4-O-benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-
a-l-rhamnopyranoside (23): Treatment of 20 (21 mg, 0.018 mmol), 1,3-
propanedithiol (0.04mL, 0.40 mmol) in pyridine (1.28 mL) and H 2O
(0.92 mL) with TEA (0.03 mL, 0.27 mmol) according to the general pro-
cedure for azide reduction and introduction of C-4’’ moitety gave free
amine (20 mg, 98%). Treatment the free amine (20 mg, 0.018 mmol) in
DMF (2 mL) with b-hydroxyisovaleric acid (4 mL, 0.037 mmol) which was
activated with HOAt (10 mg, 0.074mmol) and HATU (28 mg,
0.074mmol) in DMF (1 mL) for 1 h, and then added DIPEA (26 mL,
0.15 mmol) gave compound 23 as colorless oil (17 mg, 78%). Rf =0.61
29.5
(OCH2CH2CH2NHZ), 46.2 ((CH3)2CHCH2C(O)NH), 55.7 (C-4c), 60.3
(OCH3), 65.6 (OCH2CH2CH2NHZ), 66.5 (PhCH2OC(O)), 67.8 (C-5a),
68.5 (C-5b), 70.7 (C-5c), 72.7 (C-2a), 73.1 (C-2b), [73.3, 74.1, 75.5
(PhCH2)], 76.0 (C-3b), 78.2 (C-3a), 79.7 (C-3c), 79.8 (C-4a), 80.4 (C-4b),
84.3 (C-2c), 97.0 (C-1a), 99.2 (C-1b), 103.0 (C-1c), [127.5, 127.6, 127.7,
127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 129.7, 129.8, 129.9, 130.1,
133.0, 133.3, 136.6, 137.9, 138.3, 138.4(C arom)], 156.3 (PhCH2OC(O)),
[165.6, 165.9 (PhC(O)O)], 172.2 ((CH3)2CHCH2C(O)NH); MALDI-
TOF/MS: m/z: found: 1245.4; MALDI-FTICR/MS: m/z: calcd for
C70H82N2O17Na: 1245.5511; found: 1245.5510 [M+Na]+.
(hexane/EtOAc 1:2); 1H NMR (500 MHz, CDCl3): d 0.79 (d, 3H, J5,6
6.5 Hz, H-6c), 1.11 (s, 3H, (CH3)2C(OH)CH2C(O)NH), 1.12 (d, 3H, J5,6
=
3-[(N-Benzyloxycarbonyl)amino]propyl O-(4-acetamido-3-O-benzyl-4,6-
dideoxy-2-O-methyl-b-d-glucopyranosyl)-(1!3)-O-(2-O-benzoyl-4-O-
benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-a-l-rham-
nopyranoside (25): The azide of compound 20 was reduced as described
in the general procedures. Treatment the free amine (94mg, 0.083 mmol)
with acetic anhydride (0.016 mL, 0.17 mmol) in pyridine (0.014mL,
0.17 mmol) and DMAP (1 mg, 0.008 mmol) gave compound 25 as color-
=
6.5 Hz, H-6b), 1.14(s, 3H, (C H’3)2C(OH)CH2C(O)NH), 1.25 (d, 3H,
5,6 =5.5 Hz, H-6a), 1.74(m, 2H, OCH 2CH2CH2NHZ), 2.06 (d, 1H, J=
15.0 Hz, (CH3)2C(OH)CH2C(O)NH), 2.14(d, 1H, J=15.0 Hz,
J
(CH3)2C(OH)CH’2C(O)NH), 2.98 (m, 1H, H-5c), 3.21(m, 3H,
OCH2CH2CH2NHZ, H-3c), 3.36–3.42 (m, 3H, OCH2CH2CH2NHZ, H-2c,
H-4c), 3.52 (t, 1H, J3,4 =9.0, J4,5 =9.5 Hz, H-4b), 3.54 (t, 1H, J3,4 =9.0,
less oil (64mg, 66%) and its a-isomer (17 mg, 17%). Rf =0.25 (hexane/
1
J
4,5 =10.0 Hz, H-4a), 3.68 (m, 2H, OCH’2CH2CH2NHZ, H-5a), 3.77 (m,
EtOAc 2:3); [a]2D7 =+7.2 (c=0.4in CHCl ); H NMR (500 MHz, CDCl3):
3
1H, H-5b), 4.08 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3b), 4.14 (d, 1H, J1,2
=
d = 0.72 (d, 3H, J5,6 =6.0 Hz, H-6c), 1.12 (d, 3H, J5,6 =6.0 Hz, H-6b),
1.23 (d, 3H, J5,6 =6.5 Hz, H-6a), 1.70 (s, 3H, CH3C(O)NH), 1.73 (m, 2H,
7.5 Hz, H-1c), 4.21 (dd, 1H, J2,3 =3.0, J3,4 =9.0 Hz, H-3a), 4.49 (d, 1H, J=
11.0 Hz, PhCH2), 4.57 (d, 1H, J=11.0 Hz, PhCH’2), 4.60 (d, 1H, J=
10.5 Hz, PhCH’’2), 4.67 (d, 1H, J=11.0 Hz, PhCH’’’2), 4.73 (d, 1H, J=
11.0 Hz, PhCH’’’’2), 4.77 (s, 1H, H-1a), 4.86 (broad, 1H, NH), 4.94 (d,
1H, J=10.5 Hz, PhCH’’’’’2), 5.00 (s, 2H, PhCH2OC(O)), 5.13 (s, 1H, H-
1b), 5.32 (d, 1H, J2,3 =3.0 Hz, H-2a), 5.38 (d, 1H, J2,3 =3.5 Hz, H-2b),
5.43 (d, 1H, J=9.0 Hz, (CH3)2C(OH)CH2C(O)NH), 7.19–8.02 (m, 30H,
Harom); 13C NMR (75 MHz, CDCl3): d 17.7 (C-6c), 17.9 (C-6b), 18.1 (C-
6a), [29.3, 29.4(( CH3)2C(OH)CH2C(O)NH)], 29.7 (OCH2CH2CH2NHZ),
38.5 (OCH2CH2CH2NHZ), 47.8 ((CH3)2C(OH)CH2C(O)NH), 55.3 (C-
4c), 65.6 (OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 67.9 (C-5a), 68.7
(C-5b), 69.5 ((CH3)2C(OH)CH2C(O)NH), 70.9 (C-5c), 72.7 (C-2a), 72.8
(C-2b), [72.5, 74.6, 75.4 (PhCH2)], 74.9 (C-2c), 77.2 (C-3b), 78.0 (C-3a),
79.6 (C-3c), 80.0 (C-4a), 80.1 (C-4b), 97.1 (C-1a), 99.1 (C-1b), 103.1 (C-
1c), [127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.6, 129.7, 129.8,
130.0, 133.1, 133.3, 137.9, 138.0, 138.4(C arom)], 151.7 (PhCH2OC(O)),
[165.7, 165.9 (PhC(O)O)], 172.3 ((CH3)2C(OH)CH2C(O)NH); MALDI-
TOF/MS: m/z: calcd for C69H80N2O18Na: 1247.5304; found: 1249.7
[M+Na]+.
OCH2CH2CH2NHZ), 2.90 (m, 1H, H-5c), 2.97 (t, 1H, J1,2 =8.0, J2,3
=
8.5 Hz, H-2c), 3.12 (t, 1H, J2,3 =8.5, J3,4 =9.5 Hz, H-3c), 3.22 (m, 2H,
OCH2CH2CH2NHZ), 3.33 (t, 1H, J3,4 =9.5, J4,5 =10.0 Hz, H-4c), 3.38 (s,
3H, OCH3), 3.39 (m, 1H, OCH2CH2CH2NHZ), 3.51 (t, 1H, J3,4 =9.0,
J
4,5 =10.0 Hz, H-4b), 3.54 (t, 1H, J3,4 =9.5, J4,5 =8.5 Hz, H-4a), 3.67 (m,
2H, OCH’2CH2CH2NHZ, H-5a), 3.75 (m, 1H, H-5b), 4.13 (dd, 1H, J2,3
=
3.0, J3,4 =9.0 Hz, H-3b), 4.21 (dd, 1H, J2,3 =2.5, J3,4 =9.5 Hz, H-3a), 4.34
(d, 1H, J1,2 =8.0 Hz, H-1c), 4.48 (d, 1H, J=11.5 Hz, PhCH2), 4.53 (d, 1H,
J=11.0 Hz, PhCH’2), 4.59 (d, 1H, J=10.5 Hz, PhCH’’2), 4.70 (d, 1H, J=
12.0 Hz, PhCH’’’2), 4.77 (s, 1H, H-1a), 4.81 (d, 1H, J=11.0 Hz,
PhCH’’’’2), 4.83 (broad, 1H, NH), 4.95 (d, 1H, J=10.5 Hz, PhCH’’’’’2),
5.00 (s, 2H, PhCH2OC(O)), 5.15 (s, 1H, H-1b), 5.30 (d, 1H, J2,3 =2.5 Hz,
H-2a), 5.40 (d, 1H,
J2,3 =3.0 Hz, H-2b), 7.19–8.00 (m, 30H, Harom);
13C NMR (75 MHz, CDCl3): d = 17.5 (C-6c), 17.8 (C-6b), 18.1 (C-6a),
23.5
(CH3C(O)NH),
29.7
(OCH2CH2CH2NHZ),
(C-4c), 60.3 (OCH3),
38.5
65.6
(OCH2CH2CH2NHZ),
55.9
(OCH2CH2CH2NHZ), 66.6 (PhCH2OC(O)), 67.8 (C-5a), 68.5 (C-5b),
70.7 (C-5c), 72.8 (C-2a), 73.1 (C-2b), [73.5, 74.2, 75.5 (PhCH2)], 76.0 (C-
3b), 78.3 (C-3a), 79.6 (C-3c), 79.8 (C-4a), 80.5 (C-4b), 84.5 (C-2c), 97.0
(C-1a), 99.3 (C-1b), 103.0 (C-1c), [127.6, 127.8, 127.9, 128.0, 128.2, 128.3,
128.4, 128.5, 129.7, 129.8, 129.9, 130.2, 133.0, 133.3, 136.6, 137.9, 138.3,
138.5 (Carom)], 156.3 (PhCH2OC(O)), [165.6, 165.9 (PhC(O)O)], 169.8
(CH3C(O)NH); MALDI-TOF/MS: m/z: found: 1204.3; MALDI-FTICR/
MS: m/z: calcd for C67H76N2O17Na: 1203.5042; found: 1203.5040
[M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl O-(4-(3-methylbutanamido)-3-O-
benzyl-4,6-dideoxy-2-O-methyl-a-d-glucopyranosyl)-(1!3)-O-(2-O-ben-
zoyl-4-O-benzyl-a-l-rhamnopyranosyl)-(1!3)-2-O-benzoyl-4-O-benzyl-
a-l-rhamnopyranoside (24): The azide of compound 20 was reduced as
described in the general procedures. Treatment of the free amine (0.12 g,
0.11 mmol) in DMF (5 mL) with DIPEA (0.15 mL, 0.86 mmol) and isova-
leric acid (24 mL, 0.22 mmol) that was pre-activated with HOAt (57 mg,
0.42 mmol) and HATU (0.16 g, 0.42 mmol) in DMF (2.6 mL) for 1 h,
gave compound 24 as colorless oil (78 mg, 0.064mmol, 61%) and its a-
3-Aminopropyl O-(4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-2-O-
methyl-b-d-glucopyranosyl)-(1!3)-O-(a-l-rhamnopyranosyl)-(1!3)-a-l-
rhamnopyranoside (1): Treatment of 22 (138.0 mg, 111.3 mmol) in
isomer (19 mg, 0.016 mmol, 15%). Rf =0.39 (hexane/EtOAc 1:1); [a]D27
=
9146
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9136 – 9149

Anthrax Oligosaccharides
FULL PAPER
MeOH/CH2Cl2 (2 mL/2 mL) with NaOMe (pH 8–10) according to the
general procedure for global deprotection gave deacetylated product
(110.1 mg, 96%). Treatment of the partially deprotected compound
(110.1 mg, 106.7 mmol) in tert-butanol/H2O/AcOH (10 mL/0.25 mL/
0.25 mL) with a catalytic amount of Pd/C under an atmosphere of hydro-
gen gave compound 1 as white solid (65.5 mg, 98%). Rf =0.50 (CH3CN/
((CH3)2CHCH2C(O)NH)],
((CH3)2CHCH2C(O)NH),
22.3
26.8
(CH3COOH),
(OCH2CH2CH2NH2),
26.2
37.6
(OCH2CH2CH2NH2), 45.5 ((CH3)2CHCH2C(O)NH), 56.7 (C-4c), 60.2
(OCH3), 65.1 (OCH2CH2CH2NH2), 69.0 (C-5a), 69.4(C-5b), 70.0 (C-2a),
70.1 (C-2b), [71.0, 71.3, 71.5, 73.0 (C-4a, C-4b, C-3c, C-5c)], 78.4 (C-3a),
79.8 (C-3b), 83.5 (C-2c), 99.9 (C-1a), 102.3 (C-1b), 103.8 (C-1c), 177.2
(CH3COOH), 179.7 ((CH3)2CHCH2C(O)NH); MALDI-TOF/MS: m/z:
found: 633.2; MALDI-FTICR/MS: m/z: calcd for C27H50N2O13Na:
633.3211; found: 633.3207 [M+Na]+.
H2O/AcOH 40:20:1); 1H NMR (500 MHz, D2O): d 1.13 (d, 3H, J5,6
6.0 Hz, H-6c), 1.21 (broad, 12H, (CH3)2C(OH)CH2C(O)NH, H-6a, H-
6b), 1.92 (m, 2H, OCH2CH2CH2NH2), 2.36 (s, 2H,
=
(CH3)2C(OH)CH2C(O)NH), 3.00–3.15 (m, 4H, OCH2CH2CH2NH2, H-2c,
H-4c), 3.42–3.52 (m, 5H, OCH2CH2CH2NH2, H-4a, H-4b, H-3c, H-5c),
3.53 (s, 3H, OCH3), 3.61 (m, 1H, H-5a), 3.69–3.76 (m, 3H, H-3a, H-5b,
OCH’2CH2CH2NH2), 3.90 (d, 1H, J3,4 =10.0 Hz, H-3b), 3.93 (s, 1H, H-
2a), 4.17 (s, 1H, H-2b), 4.63 (d, 1H, J1,2 =8.0 Hz, H-1c), 4.65 (s, 1H, H-
1a), 4.93 (s, 1H, H-1b); 13C NMR (75 MHz, D2O): d [16.7, 16.8 (C-6a, C-
3-Aminopropyl O-(4-acetamido-4,6-dideoxy-2-O-methyl-b-d-glucopyra-
nosyl)-(1!3)-O-(a-l-rhamnopyranosyl)-(1!3)-a-l-rhamnopyranoside
(4): Treatment of 25 (27.2 mg, 23.0 mmol) in MeOH/CH2Cl2 (0.5 mL/
0.5 mL) with NaOMe (pH 8–10) according to the general procedure for
global deprotection gave the deacetylated product (22.9 mg, quantita-
tive). Treatment of the partially deprotected compound (22.9 mg,
23.5 mmol) in tert-butanol/H2O/AcOH (4mL/0.1 mL/0.1 mL) with a cata-
lytic amount of Pd/C under an atmosphere of hydrogen gave compound
4 as white solid (12.1 mg, 92%). Rf =0.45 (CH3CN/H2O/AcOH 40:20:1);
6b)],
17.2
(C-6c),
26.8
(OCH2CH2CH2NH2),
37.6 (OCH2CH2CH2NH2),
[28.2,
28.4
49.0
((CH3)2C(OH)CH2C(O)NH)],
((CH3)2C(OH)CH2C(O)NH), 56.7 (C-4c), 60.2 (OCH3), 65.0
(OCH2CH2CH2NH2), 68.9 (C-5a), 69.4(C-5b), 69.9
1H NMR (500 MHz, D2O): d = 1.04(d, 3H,
J5,6 =5.5 Hz, H-6c), 1.13 (d,
((CH3)2C(OH)CH2C(O)NH), 70.0 (C-2a), 70.3 (C-2b), [70.9, 71.2, 71.4,
72.9 (C-4a, C-4b, C-3c, C-5c)], 78.4 (C-3a), 79.7 (C-3b), 83.4 (C-2c), 99.8
(C-1a), 102.3 (C-1b), 103.8 (C-1c), 174.2 ((CH3)2C(OH)CH2C(O)NH);
MALDI-TOF/MS: m/z: found: 649.6; MALDI-FTICR/MS: m/z: calcd
for C27H50N2O14Na: 649.3160; found: 649.3156 [M+Na]+.
3H, J5,6 =6.5 Hz, H-6b), 1.16 (d, 3H, J5,6 =6.5 Hz, H-6a), 1.87 (s, 3H,
CH3C(O)NH), 1.89 (m, 2H, OCH2CH2CH2NH2), 2.93–3.08 (m, 4H,
OCH2CH2CH2NH2, H-2c, H-4c), 3.34–3.46 (m, 5H, OCH2CH2CH2NH2,
H-4a, H-4b, H-3c, H-5c), 3.47 (s, 3H, OCH3), 3.55 (m, 1H, H-5a), 3.64–
3.71 (m, 3H, H-3a, H-5b, OCH’2CH2CH2NHZ), 3.83 (d, 1H, J3,4
10.0 Hz, H-3b), 3.87 (s, 1H, H-2a), 4.12 (s, 1H, H-2b), 4.59 (d, 1H, J1,2
=
3-Aminopropyl O-(4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-b-d-
glucopyranosyl)-(1!3)-O-(a-l-rhamnopyranosyl)-(1!3)-a-l-rhamnopyr-
anoside (2): Treatment of 23 (17.0 mg, 13.9 mmol) in MeOH/CH2Cl2
(0.5 mL/0.5 mL) with NaOMe (pH 8–10) according to the general proce-
dure for global deprotection gave the deacetylated product (14.0 mg,
99%). Treatment of the partially deprotected compound (14.0 mg, 13.8
mmol) in tert-butanol/H2O/AcOH (2 mL/0.05 mL/0.05 mL) with a catalyt-
ic amount of Pd/C under an atmosphere of hydrogen gave compound 2
as white solid (8.1 mg, 96%). Rf =0.30 (CH3CN/H2O/AcOH 40:20:1);
1H NMR (300 MHz, D2O): d = 1.11 (d, 3H, J5,6 =6.0 Hz, H-6c), 1.17
(broad, 12H, (CH3)2C(OH)CH2C(O)NH, H-6a, H-6b), 1.84(m, 2H,
OCH2CH2CH2NH2), 2.33 (s, 2H, (CH3)2C(OH)CH2C(O)NH), 2.96 (m,
2H, OCH2CH2CH2NH2), 3.11 (t, 1H, J3,4 =7.2, J4,5 =7.2 Hz, H-4c), 3.27
=
8.0 Hz, H-1c), 4.61 (s, 1H, H-1a), 4.87 (s, 1H, H-1b); 13C NMR (75 MHz,
D2O): d
= [16.8, 17.0 (C-6a, C-6b, C-6c)], 22.3 (CH3COOH), 26.3
(CH3C(O)NH), 26.8 (OCH2CH2CH2NH2), 37.6 (OCH2CH2CH2NH2),
56.9 (C-4c), 60.2 (OCH3), 65.1 (OCH2CH2CH2NH2), 68.2 (C-5a), 69.0 (C-
5b), 69.4 (C-2a), 70.0 (C-2b), [71.0, 71.3, 71.5, 73.0 (C-4a, C-4b, C-3c, C-
5c)], 78.4(C-3a), 79.7 (C-3b), 83.3 (C-2c), 99.9 (C-1a), 102.3 (C-1b), 103.8
(C-1c), 174.8 (CH3C(O)NH), 178.4(CH 3COOH); MALDI-TOF/MS: m/
z: found: 591.2; MALDI-FTICR/MS: m/z: calcd for C24H44N2O13Na:
591.2741; found: 591.2737 [M+Na]+.
General procedure for S-acetylthioglycolylamido derivatization of the
aminopropyl spacer: The oligosaccharide 1 (10 mg, 0.016 mmol) was slur-
ried in dry DMF (500 mL) and SAMA-OPfp (7.2 mg, 0.024mmol) was
added followed by dropwise addition of DIPEA (5.6 mL, 0.032 mmol).
After stirring at room temperature for 2 h, the mixture was concentrated,
co-evaporated twice with toluene and the residue purified by size-exclu-
sion chromatography (Biogel P2 column, eluated with H2O containing
1% n-butanol) to give, after lyophilization, the corresponding thioacetate
(10.6 mg, 0.0144 mmol, 90%) as a white powder. In this manner, the thio-
acetamido derivatives of compounds 1–4 were prepared in yields of 85–
95%.
(t,
1H,
J
1,2 =7.8,
J
2,3 =8.4Hz,
H-2c),
3.36–3.60
(m,
6H,
OCH2CH2CH2NH2, H-4a, H-5a, H-4b, H-3c, H-5c), 3.64–3.75 (m, 3H, H-
3a, H-5b, OCH’2CH2CH2NH2), 3.86 (m, 2H, H-2a, H-3b), 4.14 (s, 1H, H-
2b), 4.58 (d, 1H, J1,2 =7.8 Hz, H-1c), 4.63 (s, 1H, H-1a), 4.88 (s, 1H, H-
1b); 13C NMR (75 MHz, D2O): d = [16.7, 17.2 (C-6a, C-6b, C-6c)], 27.1
(OCH2CH2CH2NH2), [28.2, 28.4(( CH3)2C(OH)CH2C(O)NH)], 37.6
(OCH2CH2CH2NH2), 49.0 ((CH3)2C(OH)CH2C(O)NH), 56.7 (C-4c), 65.1
(OCH2CH2CH2NH2),
68.9
(C-5a),
69.1
(C-5b),
69.9
((CH3)2C(OH)CH2C(O)NH), 70.0 (C-2a), 70.3 (C-2b), [71.1, 71.3, 71.4,
73.5 (C-4a, C-4b, C-3c, C-5c)], 74.2 (C-2c), 78.4 (C-3a), 79.7 (C-3b), 99.8
(C-1a), 102.3 (C-1b), 103.6 (C-1c), 174.2 ((CH3)2C(OH)CH2C(O)NH);
MALDI-TOF/MS: m/z: found: 635.3; MALDI-FTICR/MS: m/z: calcd
for C26H48N2O14Na: 635.3003; found: 635.3000 [M+Na]+.
General procedure for S-deacetylation: 7% NH3 (g) in DMF solution
(200 mL) was added to a solution of the thioacetate derivative corre-
sponding to trisaccharide 1 (2.6 mg, 3.5 mmol) in ddH2O (4 0mL) and the
mixture was stirred under argon atmosphere. The reaction was monitored
by MALDI-TOF showing the product peak of [M+Na]+. After 1 h the
solvent was evaporated under high-vacuum. The thiol derivatized trisac-
charide was further dried under high vacuum for 30 min and then used
immediately in conjugation without further purification.
3-Aminopropyl O-(4,6-dideoxy-4-(3-methylbutanamido)-2-O-methyl-b-d-
glucopyranosyl)-(1!3)-O-(a-l-rhamnopyranosyl)-(1!3)-a-l-rhamnopyr-
anoside (3): Treatment of 24 (47.0 mg, 38.4 mmol) in MeOH/CH2Cl2
(0.5 mL/0.5 mL) with NaOMe (pH 8–10) according to the general proce-
dure for global deprotection gave the deacetylated product (39.0 mg,
100%). Treatment of the partially deprotected compound (39.0 mg,
38.4 mmol) in tert-butanol/H2O/AcOH (4mL/0.1 mL/0.1 mL) with a cata-
lytic amount of Pd/C under an atmosphere of hydrogen gave compound
3 as white solid (22.1 mg, 94%). Rf =0.40 (CH3CN/H2O/AcOH 60:20:1);
1H NMR (500 MHz, D2O): d = 0.77 (m, 6H, (CH3)2CHCH2C(O)NH),
1.06 (d, 3H, J5,6 =6.0 Hz, H-6c), 1.14(m, 6H, H-6a, H-6b), 1.84(broad,
3H, OCH2CH2CH2NH2, (CH3)2CHCH2C(O)NH), 1.99 (m, 2H,
(CH3)2CHCH2C(O)NH), 2.94–2.99 (m, 4H, OCH2CH2CH2NH2, H-2c, H-
4c), 3.33–3.46 (m, 5H, OCH2CH2CH2NH2, H-4a, H-4b, H-3c, H-5c), 3.47
(s, 3H, OCH3), 3.55 (m, 1H, H-5a), 3.63 (dd, 1H, J2,3 =3.5, J3,4 =9.5 Hz,
H-3a), 3.69 (m, 2H, H-5b, OCH’2CH2CH2NHZ), 3.83 (dd, 1H, J2,3 =3.0,
General procedure for the conjugation of thiol derivatized trisaccharides
to BSA-MI: The conjugations were performed as instructed by Pierce
Endogen Inc. In short, the thiol derivative (2.5 equiv excess to available
MI-groups on the protein), deprotected just prior to conjugation as de-
scribed above, was dissolved in ddH2O (100 mL) and added to a solution
of the maleimide activated protein (2 mg) in conjugation buffer sodium
phosphate pH 7.2 containing EDTA and sodium azide (200 mL). The mix-
ture was incubated for 2 h at room temperature and then purified by Mil-
lipore Centriplus centrifugal filter devices with a 10 kDa molecular cut-
off. All centrifugations were performed at 88C for 25 min, spinning at
13ꢂg. The reaction mixture was centrifuged and the filter washed with
10 mm Hepes buffer pH 6.5 (3ꢂ200 mL). The conjugate was retrieved and
taken up in sodium phosphate buffer pH 7.4, 0.15m sodium chloride
(1 mL). This gave glycoconjugates with a carbohydrate/BSA ratio of 18:1
for trisaccharide 1, 10:1 for 2’’-OH-trisaccharide 2, 9:1 for 4’’-isovaleric
J
J
3,4 =10.0 Hz, H-3b), 3.87 (s, 1H, H-2a), 4.12 (s, 1H, H-2b), 4.57 (d, 1H,
1,2 =8.5 Hz, H-1c), 4.61 (s, 1H, H-1a), 4.86 (s, 1H, H-1b); 13C NMR
(75 MHz, D2O): d = [16.7, 16.8 (C-6a, C-6b)], 17.2 (C-6c), [21.7, 21.8
Chem. Eur. J. 2006, 12, 9136 – 9149
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9147

G.-J. Boons, C. P. Quinn et al.
acid trisaccharide 3 and 4:1 for trisaccharide 4’’-HNAc-trisaccharide 4 as
determined by Dubois’ phenol-sulfuric acid total carbohydrate assay,
quantitative monosaccharide analysis by HPAEC/PAD and Lowry pro-
tein concentration test.
was added (100 mL per well) and the incubation continued for 1 hour at
378C. Plates were then washed three times in wash buffer and 100 mL per
well of ABTS peroxidase substrate was added (KPL, Gaithersburg, MD).
Color development was stopped after 15 minutes at 378C by addition of
100 mL per well of ABTS peroxidase stop solution (KPL, Gaithersburg,
MD). Optical density (OD) values were read at a wavelength of 410 nm
(490 nm reference filter) with a MRX Revelation microtiter plate reader
(Thermo Labsystems, Franklin, MA).
Conjugation of thiol derivatized trisaccharide to KLH-BrAc: A solution
of KLH (15 mg) in 0.1m sodium phosphate buffer pH 7.2 containing
0.15m NaCl (1.5 mL) was added to a solution of SBAP (6 mg) in DMSO
(180 mL). The mixture was incubated for 2 h at room temperature and
then purified using Millipore Centriplus centrifugal filter devices with a
molecular cut-off of 30 kDa. All centrifugations were performed at 88C
for 25 min. spinning at 3000 rpm. The reaction mixture was centrifuged
off and the filter washed with conjugation buffer (2ꢂ750 mL). The acti-
vated protein was retrieved by spinning at 3000 rpm for 15 min at 88C
and taken up in 0.1 mm sodium phosphate buffer pH 8.0 containing 5 mm
EDTA (2 mL). The activated protein was added to a vial containing de-
S-acetylated trisaccharide (2.6 mg) and the mixture was incubated at
room temperature for 18 h. Purification was achieved using centrifugal
filters as described above for the BSA–MI–trisaccharide conjugates. This
gave a glycoconjugate with 1042 trisaccharide residues/KLH molecule as
determined by phenol/sulfuric acid total carbohydrate assay, quantitative
monosaccharide analysis by HPAEC/PAD and Lowry protein concentra-
tion test.
To test for competitive inhibition, the rabbit anti-live spore antiserum or
the rabbit anti-irradiated spore antiserum was added together with un-
conjugated trisaccharide in blocking solution at a 6-, 12-, 25-, 50-, 100-, or
200-fold weight excess compared to weight of carbohydrate used for
coating. The negative control consisted of uncoated wells incubated with
the respective antiserum plus trisaccharide 1 at a concentration corre-
sponding to “200-fold excess” of trisaccharide.
To explore competitive inhibition by synthetic saccharide analogues con-
jugated to bovine serum albumin (BSA; Pierce Biotechnology, Rockford,
IL), rabbit anti-live spore antiserum was diluted 1:1600 in blocking solu-
tion. For each well 100 mL of the serum were mixed with either 100 mL
blocking solution or 100 mL of BSA-MI-conjugate in blocking solution
with a concentration corresponding to a 2-, 4-, 8-, 16-, 32-, 64-, or 128-
fold weight excess of carbohydrate compared to carbohydrate used for
coating. The four conjugates tested were: BSA-MI-1, BSA-MI-2, BSA-
MI-3, and BSA-MI-4. First the serum and then the BSA–saccharide con-
jugate solutions were added to an uncoated microtiter plate and mixed
by pipetting up and down before the well contents were transferred to a
coated plate. The microtiter plates were incubated and developed as de-
scribed above.
Preparation of Bacillus anthracis Sterne 34F2 spores: Bacillus anthracis
Sterne 34F2 was obtained from the CDC culture collection. Spores of B.
anthracis Sterne 34F2 were prepared from liquid cultures of PA
medium[41] grown at 378C, 200 rpm for six days. Spores were washed two
times by centrifugation at 10000ꢂg in cold (48C) sterile deionized water,
purified in a 50% Reno-60 (Bracco Diagnostics Inc., Princeton, NJ) gra-
dient (10000ꢂg, 30 min, 48C) and washed a further four times in cold
sterile deionized water. After suspension in sterile deionized water,
spores were quantified with surface spread viable cell counts on brain
heart infusion (BHI) agar plates (BD BBL, Sparks, MD). Spore suspen-
sions were stored in water at ꢀ808C.
The data are reported as the means ꢁSD of triplicate measurements.
For the preparation of killed spores, 500-mL aliquots of spore suspensions
in water, prepared as described above and containing approximately 3ꢂ
108 CFU, were irradiated in 2.0 mL Sarstedt freezer tubes (Sarstedt,
Newton, NC) in a gammacell irradiator with an absorbed dose of 2 mil-
lion rads. Sterility after irradiation was monitored by spread-plating 10-
mL aliquots of irradiated spore suspension on BHI agar plates. The plates
were incubated for 72 h at 378C and monitored for colony growth. Ab-
sence of growth was taken as an indicator of sterility.
Acknowledgements
This work was supported by NIAID Grant R21 AI059577 from the Na-
tional Institutes of Health.
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microtiter plates (Thermo Labsystems, Franklin, MA) were coated with
100 mL per well of the KLH-BrAc-1 conjugate at a concentration of
0.03 mg per mL of carbohydrate content, corresponding to 0.5 mg per mL
by protein content, or by the protein mcKLH by itself (0.5 mg per mL
protein) in coating buffer (0.01m PBS, pH 7.4). Plates were washed three
times in wash buffer (0.01m PBS, pH 7.4, 0.1% Tween-20) using an
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Received: August 31, 2006
Chem. Eur. J. 2006, 12, 9136 – 9149
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