Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens

Article abstract of DOI:10.1021/jo300299p

Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide

Full text of DOI:10.1021/jo300299p

Article
pubs.acs.org/joc
Synthetic Oligosaccharides as Tools to Demonstrate Cross-Reactivity
between Polysaccharide Antigens
Vince Pozsgay,* Joanna Kubler-Kielb, Bruce Coxon, Paul Santacroce, John B. Robbins,
and Rachel Schneerson
Program on Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892-2423, United States
S
* Supporting Information
ABSTRACT: Escherichia coli O148 is a nonencapsulated
enterotoxigenic (ETEC) Gram negative bacterium that can
cause diarrhea, hemorrhagic colitis, and hemolytic uremic
syndrome in humans. The surface-exposed O-specific
polysaccharide (O-SP) of the lipopolysaccharide of this
bacterium is considered both a virulence factor and a
protective antigen. It is built up of the linear tetrasaccharide
repeating unit [3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-
GlcNAcp-(1→3)-α-L-Rhap-(1→] differing from that of the
O-SP of Shigella dysenteriae type 1 (SD) only in that the latter
contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria
indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-
SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of
mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the
LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-
blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.
unit: the galactose residue in SD is replaced by a D-glucose moiety
in the E. coli repeating unit, while the anomeric configurations
and the locations of all of the interglycosidic linkages are
preserved. The two bacteria have the same genes for O antigen
synthesis, except that in SD, a glucosyltransferase gene is
interrupted by a deletion, and a galactosyltransferase gene
located on a plasmid is responsible for the transfer of galactose to
synthesize the O-antigen.8 The two bacteria also have chemically
identical LPS cores and O-chain−core linkage regions.10
On the basis of the close similarity of the O-SPs of E. coli O148
and SD, we hypothesized that they may cross-react; i.e.,
antibodies raised against one of the O-SPs or their fragments
will react (bind) not only with the saccharides of the homologous
organism but also with those of the cross-reacting ones. We note,
however, that cross-reactivity between similar surface poly-
saccharides of different bacteria is not obvious. We approached
this question by using synthetic oligosaccharides.
INTRODUCTION
■
Escherichia coli O148 is an enterotoxigenic (ETEC) bacterium
that has been identified as a cause of enteric infections in children
and adults, including dysentery, hemorrhagic colitis, and
hemolytic uremic syndrome. This disease is endemic in
developing countries and affects travelers to those areas.1−5
The main carriers of ETEC bacteria are food and water,5 and
their spreading is facilitated by poor sanitary conditions. As has
been reported recently regarding the spread of the bacterium E.
coli O104 in several Western European countries, even the best
sanitary conditions cannot always prevent an epidemic. It is likely
that infections caused by various ETEC serotypes are under-
reported because of insufficient surveillance and difficulties in
serotyping. In early 2009, the World Health Organization
declared the development of a vaccine against ETEC an
urgency.6,7
We have reported the synthesis of a panel of oligosaccharides
related to the O-SP of SD up to a tetracosasaccharide11−13 and
demonstrated that the immunogenicity of their BSA conjugates
in mice is influenced by the size of the saccharides, their loading
on the protein, as well as by the identity of the nonreducing
terminus.12,14
E. coli O148 has been proposed to be a precursor to Shigella
dysenteriae type 1 (SD), the most virulent of all Shigellae.8
Virulence of these two bacteria is related to their O-specific
polysaccharide (O-SP): those that lack a fully developed O-SP
are not considered virulent.9 Of the two, SD is the more common
pathogen. The repeating unit of the O-SP of E. coli O148 A is
similar to the O-SP of SD B, the only difference being in the
chirality of a single carbon atom in the tetrasaccharide repeating
Received: February 27, 2012
© XXXX American Chemical Society
A
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Figure 1. Spacer-equipped oligosaccharide fragments of the O-SP of E. coli O148 synthesized in this study.
In this paper, we report our studies that may lead to the
development of a single component neoglycoconjugate vaccine
against the enteric bacteria SD and E. coli O148, consisting of a
covalent conjugate of an oligosaccharide fragment of the O-
specific oligosaccharide of only one of them, covalently attached
to an immunogenic protein. The idea behind using a protein
conjugate of synthetic or natural oligosaccharides to induce
anticarbohydrate serum is not new: it dates back to the early part
of the twentieth century when it was shown that a covalent
conjugate of the capsular polysaccharide of Type 3 pneumo-
B
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Figure 2. Retrosynthetic strategy toward the targeted oligosaccharides.
coccus with horse serum globulin elicited antipolysaccharide-
specific antibodies in rabbits.15−17 The antisera conferred both
active and passive protection against the homologous organism.
On the basis of this idea, several polysaccharide−protein
conjugate vaccines have been developed for human use,
including vaccines against Haemophilus influenzae type b,
Neisseria meningitidis serotypes A, C, Y, W-135, pneumococci,
and Salmonella typhi.18 A tetanus toxoid conjugate of synthetic
fragments of the capsular material of H. influenzae type b is also
an efficacious vaccine for both children and adults.19 The
potential of synthetic oligosaccharide fragments of bacterial cell-
surface glycans as antibacterial vaccines has generated increasing
interest in the field that led to the synthesis of numerous bacterial
oligosaccharides20−24 and improved conjugation methods.25
Here, we first report the chemical synthesis of oligosaccharide
components 1−7 of the O-SP of E. coli O148, ranging from tetra-
to dodecasaccharides (Figure 1), then test their immunogenicity
and cross-reactivity with the O-SP of SD. Oligosaccharides 1−7
are equipped with a heterobifunctional spacer to allow their one-
point conjugation to the proteins bovine serum albumin (BSA)
and recombinant diphtheria toxin H21G.26 This protein is a
genetically modified diphtheria toxin in which a histidine residue
is replaced by glycine. The toxicity of the resulting protein is
greatly reduced,27 and the recombinant diphtheria toxin H21G
has been shown to be an immunogenic carrier suitable for the
preparation of conjugate vaccines.28
These constructs, injected without an adjuvant at a schedule
and dosage compatible with use for humans, were used to
evaluate the antibody response to the native O-SPs in mice. We
C
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a
Scheme 1. Synthesis of Glucosyl Trichloroacetimidate 11
a
Reagents and conditions: (a) NaOMe, MeOH, quant.; (b) BnBr, NaH, DMF, 95%; (c) AcOSi(CH3)3 (excess), reflux, 2 h, 93%; (d) PhSSi(CH3)3,
BF3, Et2O, CH2Cl2, 0 °C, 90 min, 84%; (e) MeONa, MeOH, 4 h, 97%; (f) PMBCl, NaH, DMF, 91%; (g) (CF3CO2)2Hg, CH2Cl2, H2O, 97%; (h)
CCl3CN, Cs2CO3, CH2Cl2, quantitative.
a
Scheme 2. Synthesis of the Trisaccharide Trichloroacetimidate 28
a
Reagents and conditions: (a) 1.6 equiv of 11, TMSOTf, −40 → 0 °C, 2 h, 90%; (b) H2 (200 psi), Pd/C, Et3N, EtOAc, EtOH, Ac2O; (c)
Ce(NH4)2(NO3)6, CH3CN, H2O, yields: 57% for 10, 11% for 25; (d) Ac2O, C5H5N, DMAP, CH2Cl2, quantitative; (e) TFA, CH2Cl2, 80%; (f)
CCl3CN, DBU, CH2Cl2, 95%.
also describe the isolation of purified LPSs of E. coli O148 and SD
and report on their binding to mice sera elicited by various
protein conjugates of the synthetic oligosaccharides, and to sera
generated in rabbits by heat-killed E. coli O148 and SD. The
selection of this panel of oligosaccharides was based on our
observations on the immunogenicity of BSA conjugates of SD-
D
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a
Scheme 3. Synthesis of the Tetrasaccharide Trichloroacetimidate 8
a
Reagents and conditions: (a) 4.5 equiv of 9, TMSOTf, CH2Cl2, 0 → 23 °C, 3 h, 91%; (b) TFA, CH2Cl2, 23 °C, 3 h, 65%; (c) CCl3CN, DBU,
CH2Cl2, 0 → 23 °C, 1 h, 78%.
related oligosaccharides in mice.12,14 In those studies, we found
that the conjugates containing an N-acetyl-D-glucosamine or a D-
galactose residue at the nonreducing end of the oligosaccharide
portion induced statistically significantly higher O-SP-specific
IgG antibody levels in mice than those having an L-rhamnose
moiety at that position.
It is to be expected that mapping the immunogenicity of the
protein conjugates of the synthesized oligosaccharides will
contribute to the development of oligosaccharide-based semi-
synthetic vaccines against enteric diseases.
solution of orthoester 17 in AcOSi(CH3)3 afforded the diacetate
18 in 93% yield after chromatographic purification. Next, 18 was
converted to thioglucoside 19 with trimethylsilylthiophenol34 in
the presence of BF3·Et2O in 84% yield. Subsequently, the acetyl
group in 19 was removed by NaOMe in MeOH to afford the
alcohol 20. The β anomeric configuration was ascertained from
the J1,2 coupling constant whose value was 9.5 Hz. Next,
compound 20 was reacted with 4-methoxybenzyl chloride in the
presence of NaH to afford 21 in 91% yield. Subsequent
hydrolysis of the thioglucosyl linkage by the action of
(CF3CO2)2Hg11,35 in moist CH2Cl2 afforded the hemiacetal 22
in 97% yield, from which the Schmidt-type donor 11 was
prepared in nearly quantitative yield by treatment with CCl3CN/
Cs2CO3.
With the glucosyl imidate 11 in hand, synthesis of the
trisaccharide donor 28 was undertaken. (Scheme 2) First, the
disaccharide alcohol 1229 was glucosylated with compound 11
using TMSOTf activation. Under these conditions, an
inseparable 4:1 mixture of the α and β-linked trisaccharides 23
was obtained in a combined yield of 90%. Attempted activation
of 11 by other Lewis acids, e.g., BF3-etherate, failed to improve
the anomeric stereoselectivity. Next, the azido group was
converted to acetamido by catalytic hydrogenation over
palladium-on-charcoal in the presence of Et3N, followed by
reaction with Ac2O (→ 24). Subsequent treatment with
ammonium ceric nitrate in a mixture of CH3CN and water
removed the methoxybenzyl group. To our delight, the required
trisaccharide alcohol 10 could be isolated in a pure form in 57%
yield after column chromatographic purification without the β
glucosyl-linked minor product 25 that was also recovered in 11%
yield. The synthesis of the trisaccharide 28 was completed by (i)
acetylation of HO-2 of the glucose residue in 10 (→ 26,
quantitative), (ii) removal of the trimethylsilylethyl group by
treatment of compound 26 with trifluoroacetic acid36 (→ 27,
80%), and (iii) exposure of the hemiacetal 27 to CCl3CN/DBU
to afford the trisaccharide trichloroacetimidate 28 in 95% yield.
The tetrasaccharide building block 8 was synthesized by
coupling the trisaccharide acceptor 10 with the rhamnosyl donor
911 under TMSOTf activation, to afford tetrasaccharide 29 in
RESULTS
■
Synthetic Studies. We envisioned a blockwise approach to
the target oligosaccharides 1−7 that required mono-, di-, tri-, and
tetrasaccharide building units (Figure 2). For example, the
retrosynthetic analysis of the dodecasaccharide 7 called for the
tetrasaccharide donor 8 as the crucial intermediate that was
prepared from the rhamnosyl donor 911 and the trisaccharide
acceptor 10 that in turn was constructed by condensation of the
glucosyl donor 11 and the disaccharide alcohol 12.29 The spacer-
linked tetrasaccharide 13 was obtained from the imidate 8. The
synthesis of the dodecasaccharide 14 was completed by two
iterative sequential chain extension steps using the tetrasacchar-
ide donor 8. This was followed by global deprotection and
installation of the linker to afford 7, in which the spacer features a
keto functionality to be used for the conjugation to proteins,
using the oxime methodology.30 The syntheses of the
oligosaccharides 1−6 were carried out in a similar fashion, as
described in detail below.
The tetrasaccharide block 8 was prepared from the
trisaccharide 10, assembly of which required the D-glucose
derivative 11 that was prepared as shown in Scheme 1. The
starting material was orthoester 15 that was deacetylated (→ 16)
followed by O-benzylation to afford compound 17.31 Next, we
attempted to convert orthoester 17 into the diacetate 18, using
acetic acid.32 It has been noted that in such conversions the acetic
acid must be meticulously dry, because even traces of water
would lead to complete hydrolysis.31,32 A safer alternative is the
use of AcOSi(CH3)3 instead of acetic acid.33 Thus, boiling a
E
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a
Scheme 4. Synthesis of the Spacer-Linked Tetrasaccharide Acceptor 32
a
Reagents and conditions: (a) 1.4 equiv of 31, TMSOTf, CH2Cl2, 0 °C, 1 h, 81%; (b) CS(NH2)2, C5H5N, DMF, 23 °C, 12 h, 83%.
91% yield (Scheme 3). Routine removal of the trimethylsilylethyl
group with trifluoroacetic acid in CH2Cl2 (→ 30, 65%)
chemical shifts are shown for 41 and 42 in Table 5, and for 14 in
Table 6. Finally, values of the JC‑1,H‑1 coupling constants
36
1
followed by reaction with CCl3CN/DBU yielded the tetrasac-
charide trichloroacetimidate 8 in 78% yield.
measured by 1H-coupled 2D HSQC for 41, 42, and 14 may be
compared in Table 7. The observation of exclusively large 1JC‑1,H‑1
values (168.9−175.8 Hz) by 1H-coupled 2D HSQC indicates the
α anomeric configuration for all linkages in the three
oligosaccharide glycosides, as synthetically designed. Similar
repeating structural environments in glycosides 42 and 14 cause
equivalence or near-equivalence of the chemical shifts of residues
in the same immediate environment. These residues are Glc C
and G in 42, and residues Glc C, G and K; GlcNAc F and J; Rha D
and H; and Rha E and I, in 14. For example, the multiplet
patterns in the 1H-coupled 2D HSQC spectra of 42 (Figure 4a)
and 14 (Figure 4b) are remarkably similar. However, close
inspection of these spectra reveals intensity differences due to the
coincidence of resonances of residues in equivalent environ-
ments. According to the 1H chemical shifts of 14 (Table 2), the
structural environment of GlcNAc B is observably different to
those of GlcNAc F and J, apparently because of the proximity of
GlcNAc B to the unique Rha A residue at the glycoside terminus.
Characteristic 13C-1 chemical shifts were observed for the
different types of residues: GlcNAc at ∼95 ppm, Glc at 98.5 ppm,
Rha A at ∼100 ppm, Rha glycosidically attached to Glc at 102.1−
102.3 ppm, and Rha glycosidically bound to Rha at 102.7−102.8
ppm. All of the linkage positions in the three oligosaccharide
glycosides 41, 42, and 14 were confirmed by the observation of
specific connectivities in the 2D HMBC spectra, for one or two
As a prelude to the assembly of higher-membered
oligosaccharides, the tetrasaccharide donor 8 was condensed
with methoxycarbonylpentanol 3137 under TMSOTf promo-
tion, to afford compound 13 in 81% yield (Scheme 4). We favor
aglycon 31 as the linker because it is stable under a variety of
conditions and can easily be converted to several reactive species
for incorporation into proteins. Routine removal of the
monochloroacetyl group by thiourea38 afforded the tetrasac-
charide alcohol 32 that was subjected to further chain extension
steps with glycosyl donors as presented in Scheme 5.
Condensation of the alcohol 32 with the di- 33,29 tri- (28),
and tetrasaccharide (8) donors using TMSOTf as the activator in
CH2Cl2 afforded the fully protected hexa- (34), hepta- (35), and
octasaccharides (36) in the range of 60 to 91% yields. For further
chain extension, the temporary monochloroacetyl group was
removed from the fully protected octasaccharide 36 by treatment
with thiourea to afford the alcohol 37 (77%). Glycosylation of
the octamer 37 with the di- (33), tri- (28), and tetrasaccharides
(8) using TMSOTf as the activator yielded the deca- (38),
undeca- (39), and dodecasaccharides (40) (Scheme 5). In these
condensations, the donors were used in 2- to 3-fold molar
excesses, and the isolated yields were in the 60−91% range.
Although the isolated yields of the protected oligosaccharides
were, in most cases, acceptable, the procedures were not without
the need for repeated column chromatographic purification,
steps that undoubtedly contribute to the often moderate yields.
Preparation of the targets 1−7 from the fully protected tetra-
to dodecasaccharides 13, 34, 35, 36, and 38−40 (denoted C in
Scheme 6) proceeded in four stages: (i) base-promoted removal
of the O-acyl protecting groups, (ii) hydrogenolytic removal of
the O-benzyl groups (→ D), (iii) aminolysis with ethylenedi-
amine (→ E), and (iv) N-acylation with 5-ketohexanoic
anhydride (→ F). Removal of all the protecting groups was
ascertained by the 1H and 13C NMR spectra of the
oligosaccharides so obtained that were consistent with the
proposed structures.
1
13C,H pairs per linkage. Interpretation of the H−1H coupling
constants (Tables 3 and 4) for the glycosides 41, 42, and 14
according to Karplus considerations confirmed the types of sugar
residues present and their chair conformations.
Conjugation of the Oligosaccharides to Proteins.
Oligosaccharides do not elicit an immune response when
injected into mammals. However, they can be converted to
immunogens when covalently linked to immunogenic proteins.
Of the numerous possible approaches, selective oxime formation
between a keto-derivatized carbohydrate and an aminooxy-
equipped protein counterpart stands out with its simple
operation, mild conditions, and applicability to a variety of
saccharide haptens, ranging from mono- to polysacchar-
ides.14,30,39 In our approach (Scheme 7), the spacer-equipped
oligosaccharides 1−7 (F) were combined with the aminooxy-
derivatized proteins I to afford the conjugates J, at pH 7.2 or
below.
The aminooxy moiety was introduced into the protein carriers
in a two-step process. First, the proteins BSA or recombinant
diphtheria toxin (G) were reacted with succinimidyl (3-
bromoacetamido)propionate to afford structure H featuring a
labile bromine moiety that, upon reacting with 2-amino-
oxypropanethiol,40 allowed the formation of a stable thioether
linkage connecting the derivatized protein and the aminooxy
In addition to further transformations as described below,
three representative intermediates 14, 41, and 42 (Figure 3) were
used in detailed NMR studies.
Nuclear Magnetic Resonance Studies. The three
methoxycarbonylpentyl glycosides 14, 41, and 42 were examined
in detail by 1H and 13C NMR spectroscopy, using 2D COSY-30
and TOCSY to assign the 1H spectra, and 2D HSQC and HMBC
to confirm assignments for the 13C spectra. 1H chemical shifts for
the glycosides 41 and 42 are shown in Table 1, and for 14 in
1
Table 2. Vicinal and geminal H−1H coupling constants are
listed for 41 and 42 in Table 3, and for 14 in Table 4. 13C
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aminooxy moieties were attached to the proteins. In order to
avoid overderivatization that might reduce immunogenicity, the
number of the carbohydrate chains were limited to an average of
20 or less. The conjugates were purified through a Sephadex G-
50 column to afford neoglycoproteins having an average of up to
15 oligosaccharide chains, as determined by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS). For the details of the determination of
the average number of carbohydrate chains, see ref 30.
Scheme 5. Synthesis of the Higher-Membered
Oligosaccharides 34−40
a
Serum Antibody Responses. The immunogenicity of the
conjugates containing the E. coli O148 oligosaccharides and
those made from oligosaccharide portions related to the LPS of
SD14 (Table 8) was estimated in groups of 10 mice (Table 9).
Three subcutaneous injections containing 2.5 μg of saccharides
in the form of conjugates in phosphate buffered saline (PBS)
were given without adjuvant at 2 week intervals. We note that
almost all of the synthetic oligosaccharide-based carbohydrate−
protein conjugates reported in the literature have been
administered in Freund’s complete adjuvant followed by two
injections in Freund’s incomplete adjuvant. Since Freund’s
adjuvant cannot be given to humans, the extrapolation of the
experimental data for human use is at least questionable. All of
the conjugates tested induced significantly higher IgG antibody
levels against both LPSs than the control group that received PBS
at the same schedule (not shown, p < 0.0001). There were no
statistical differences between the anti-SD IgG levels induced
either by the SD or by the E. coli oligosaccharide conjugates (data
in column 5, Table 9) with one exception: the conjugate of the E.
coli 10mer 5 (item no. 5) induced a statistically lower anti-SD
level than did the conjugate of the SD 10mer (44) conjugate
(item no. 11, 4.6 vs 11.8 EU, p = 0.03). Similarly, there were no
statistical differences between the anti-E. coli O148 levels induced
by any of the conjugates (column 6, Table 9). The highest
antibody levels against both LPSs were induced by the BSA
conjugate of the E. coli octasaccharide 4 (item no. 4). Some of the
differences, e.g., the higher anti-SD response by the conjugate of
the E. coli 8-mer 4 versus that of the SD octasaccharide 43 (item
10), may be a consequence of different loadings on the protein.
In general, the conjugates that induced high anti-SD IgG levels
also induced high anti-E. coli O148 IgG levels. These data
support the assumption that immunity to either of these bacteria
would protect the host against both of them.
Immunoblotting. Western blot assays were performed to
evaluate if sera of immunized animals cross-react with LPSs of
both bacteria. Sera used in these experiments were induced either
in rabbits by heat-killed bacteria or in mice by the conjugates of
synthetic oligosaccharides corresponding to their O-SP subunits:
compound 7 (12mer) in the case of E. coli O148 and compounds
44, 45, 46, and 47 (10, 11, 12, and 13mer) (Table 9) in the case
of SD.14 The results presented in Figure 5 show that all sera
reacted with both LPSs with similar intensity and in both
directions.
In a dot-blot experiment, BSA conjugates of the mono-
saccharides galactose and glucose containing an average of 22
monosaccharide residues per protein (not described in the
experimental) did not bind to sera raised to either bacterium.
The BSA conjugate of compound 1 (4mer), representing one
repeating unit of E. coli O148 O-SP, bound to sera raised by
either bacterium less strongly than did conjugates of compounds
2−7, which underwent binding with approximately the same
intensity.
a
Reagents and conditions: (a) 2.3 equiv of 33, TMSOTf, CH2Cl2, 0
°C, 45 min, 91%; (b) 2.3 equiv of 28, TMSOTf, CH2Cl2, 0 °C, 40 min,
60%; (c) 2.1 equiv of 8, TMSOTf, CH2Cl2, 0 → 23 °C, 1 h, 88%; (d)
CS(NH2)2, C5H5N, DMF, 23 °C, 16 h, 77%; (e) 5.2 equiv of 33,
TMSOTf, CH2Cl2, 0 → 23 °C, 1 h, 88%; (f) 3.5 equiv of 28,
TMSOTf, CH2Cl2, 0 → 23 °C, 1 h, 86%; (g) 1.9 equiv of 8, TMSOTf,
CH2Cl2, 0 → 23 °C, 1 h, 64%.
moiety (→ I). Depending on the ratio of the bromoacyl
derivatized protein and the aminooxypropanethiol, up to 35
Competitive Inhibition of Binding. In order to corrobo-
rate the results of the immunoblotting experiments and
G
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Scheme 6. Removal of the Protecting Groups and Attachment of the Linker Moiety
Figure 3. Tetra- 41, octa- (42), and dodecasaccharide (14) methoxycarbonylpentyl glycosides.
demonstrate further that the cross-reactivity between SD and E.
coli O148 is due to the O-SP portion of their respective
lipopolysaccharides, we analyzed the inhibition of binding
between sera induced by DT conjugates of SD 12mer
(compound 46) and E. coli O148 12mer (compound 7) and
their respective LPSs (Tables 10 and 11), using the O-SP of the
unrelated bacterium Bordetella bronchiseptica as the control. The
data in Tables 10 and 11 demonstrate dose-related inhibition of
binding within similar ranges, thus confirming that the cross
reactivity between SD and E. coli O148 is, indeed, due to their
respective O-SPs.
DISCUSSION
■
As part of our program directed toward the development of
carbohydrate-based conjugate vaccines against human patho-
genic bacteria, we were intrigued as to whether there exists cross-
H
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Table 1. 1H NMR Chemical Shifts (ppm) of the E. coli O148 Tetra- (41) and Octasaccharide (42) 5-Methoxycarbonylpentyl
Glycosides
tetrasaccharide 41
octasaccharide 42
A
B
C
D
MeOCO
Pentyl
A
B
C
D
E
F
G
H
MeOCO
Pentyl
Rha
4.806
GlcNAc
Glc
Rha
5.106
Rha
4.801
GlcNAc
Glc
Rha
5.103
Rha
5.123
GlcNAc
Glc
Rha
5.127
H-1
H-1′
H-2
H-3
H-4
H-5
H-6
H-6′
MeO
NAc
4.981
−
5.534
3.702
3.527
1.627
1.377
1.627
2.404
−
4.980
−
5.564
5.038
−
5.564
3.695
3.512
1.612
1.377
1.612
2.391
−
−
−
−
−
−
−
−
−
−
4.067
3.810
3.525
3.722
1.312
4.128
4.032
3.785
∼4.012
3.808
3.808
−
3.660
3.730
3.493
∼3.639
3.852
3.802
−
4.069
3.788
3.471
3.840
1.293
4.066
3.825
3.547
3.717
1.314
4.136
4.067
3.803
∼4.041
3.841
3.841
−
3.662
3.754
3.488
3.658
3.876
3.796
4.182
3.886
3.572
3.901
1.303
4.247
3.968
3.576
3.915
1.349
4.140
4.081
3.794
∼4.053
3.845
3.804
−
3.662
3.754
3.486
3.658
3.868
3.796
4.077
3.802
3.490
3.857
1.303
−
−
−
−
−
−
−
3.690
−
−
−
−
−
−
−
−
−
−
−
−
−
3.685
−
−
−
−
a
a
2.041
−
2.053
2.061
−
a
Interchangeable.
Table 2. 1H NMR Chemical Shifts (ppm) of the E. coli Dodecasaccharide 5-Methoxycarbonylpentyl Glycoside 14
dodecasaccharide residues
A
B
C
D
E
F
G
H
I
J
K
L
MeOCO
Pentyl
Rha
4.801
GlcNAc
Glc
Rha
Rha
GlcNAc
Glc
Rha
Rha
5.136
GlcNAc
Glc
Rha
5.139
H-1
H-1′
H-2
H-3
H-4
H-5
H-6
H-6′
MeO
NAc
4.980
−
5.581
5.121
−
5.136
5.044
−
5.580
5.118
−
5.044
−
5.578
3.692
3.506
1.612
1.380
1.612
2.386
−
−
−
−
−
−
−
−
a
a
4.065
3.833
3.560
3.713
1.315
4.145
4.088
3.811
4.052
3.838
3.814
−
3.662
3.767
3.483
3.661
3.890
3.793
4.191
3.896
3.586
∼3.905
1.311
−
4.258
3.987
3.590
3.928
1.356
4.150
4.102
3.797
4.064
3.860
3.804
−
3.662
3.767
3.483
3.661
3.890
3.793
4.189
3.895
3.585
∼3.904
1.308
−
4.261
3.987
3.590
3.590
1.356
4.150
4.102
3.797
4.064
3.860
3.804
−
3.662
3.767
3.481
3.661
3.881
3.793
4.083
3.810
3.501
3.865
1.308
−
−
−
−
−
−
−
−
−
−
−
−
−
3.684
−
−
−
−
−
−
−
−
−
−
b
b
b
2.061
2.072
2.070
−
a,b
Interchangeable within each letter group.
Table 3. 1H−1H Coupling Constants (Hz) of the E. coli O148 Tetra- (41) and Octasaccharide (42) 5-Methoxycarbonylpentyl
Glycosides
tetrasaccharide residues
octasaccharide residues
A
B
C
D
A
B
C
D
E
F
G
H
Rha
GlcNAc
Glc
Rha
Rha
GlcNAc
Glc
Rha
Rha
GlcNAc
Glc
Rha
J1,2
J2,3
J3,4
J4,5
J5,6
J5,6′
J6,6′
2.1
3.3
10.1
9.8
6.3
−
3.6
10.6
8.8
3.6
10.0
8.9
1.8
3.6
10.0
9.8
6.3
−
1.7
3.6
10.2
9.8
6.2
−
3.6
10.7
8.7
10.0
nr
3.6
9.8
1.7
3.2
9.6
9.4
6.2
−
∼2.1
3.3
10.6
9.7
6.2
−
3.6
10.7
8.7
3.6
9.8
1.8
3.6
10.1
9.8
6.4
−
9.1
8.8
10.2
10.1
2.3
10.2
2.3
9.8
10.0
2.1
a
nr
2.0
nr
nr
4.2
nr
4.6
3.9
4.6
−
12.3
−
−
nr
12.2
−
−
12.4
12.0
−
a
nr, not resolved.
reactivity between the O-SPs of SD and E. coli O148 based on
their close structural similarity. We hypothesized that cross
reactivity between the O-SPs may serve as a basis for protection
against both bacteria by a single monovalent vaccine and decided
to explore cross-reactivity by using oligosaccharide fragments of
the O-SPs of both bacteria, in the form of covalent conjugates
with immunogenic proteins. Such oligosaccharides are difficult to
isolate in homogeneous form from the native O-SP because of
the difficulty of its site-specific fragmentation by either chemical
or enzymatic processes. Therefore, we prepared the required
oligosaccharides by chemical syntheses as described in detail in
the Experimental Section.
The synthetic oligosaccharides were equipped with a linking
arm featuring an oxo function for covalent attachment to
immunogenic proteins, including bovine serum albumin and
recombinant diphtheria toxin, by using the oxime method.30 The
level of incorporation was determined by MALDI-TOF mass
spectrometry and reached up to an average of 15 oligosaccharide
chains per protein molecule. The neoglycoconjugates, subcuta-
neously injected in groups of 10 mice, induced IgG antibodies
with O-SP specificities shown by immunochemical experiments.
Western-blot analysis demonstrated that sera raised against
heat-killed SD or E. coli O148 bacteria in rabbits or against the
synthetic oligosaccharide−protein conjugates in mice reacted
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Table 4. 1H−1H Coupling Constants (Hz) of the E. coli O148 Dodecasaccharide 5-Methoxycarbonylpentyl Glycoside 14
dodecasaccharide residues
A
B
C
D
E
F
G
H
I
J
K
L
Rha
GlcNAc
Glc
Rha
Rha
GlcNAc
Glc
Rha
Rha
GlcNAc
Glc
Rha
J1,2
J2,3
J3,4
J4,5
J5,6
J5,6′
J6,6′
1.8
3.1
9.9
9.6
6.2
−
3.6
10.6
8.5
3.8
10.0
9.1
1.8
3.1
9.7
9.6
6.2
−
1.5
3.4
9.9
9.7
6.2
−
3.6
10.5
8.6
3.7
10.0
9.1
1.7
3.1
9.8
9.7
6.3
−
2.0
3.3
9.9
9.7
6.2
−
3.6
10.5
8.6
3.7
10.0
9.1
1.8
3.3
10.1
9.7
6.2
−
10.2
3.0
10.0
2.2
9.9
10.0
2.2
9.9
10.1
2.1
2.5
2.5
4.3
5.1
4.9
5.1
4.9
5.1
−
12.8
12.3
−
−
∼11.7
12.3
−
−
∼11.7
12.2
−
General Procedure for the Deprotection of Oligosaccharides
13, 34−36, and 38−40. The protected oligosaccharide C (Scheme 6)
was dissolved in anhydrous CHCl3 to which anhydrous MeOH was
added. To the resulting solution was added a solution of NaOMe in
MeOH (25 wt %) at room temperature until the pH of the solution
reached approximately 12 as estimated with a moistened indicator
paper. After 24 h, the solution was neutralized with Dowex (H+) resin.
Next, the resin was removed by filtration, and the volatiles were removed
under reduced pressure. To a solution of the residue so obtained in
EtOH was added Pd/C, and the resulting mixture was stirred under
hydrogen (200 psi) for 24 h. Removal of the catalyst by filtration
through a layer of Celite followed by concentration afforded the
deprotected oligosaccharides as 5-methoxycarbonyl glycosides D. In
addition to further transformations as described below, compounds 14,
41, and 42 (Figure 3) were used in NMR studies.
General Procedure for the Preparation of the Spacer-Linked
Oligosaccharides 1−7. The deprotected oligosaccharide was
dissolved in 1,2-diaminoethane. After 24 h at room temperature, the
solution was diluted with water followed by evaporation of the volatiles
by freeze-drying. The residue was purified by chromatography through a
Bio-Gel P-2 column, using 0.02 M pyridine acetate in water as the eluant.
The fractions containing carbohydrates as determined by the phenol-
sulfuric acid assay were combined, and the solution was freeze-dried.
This cycle was repeated two more times to afford intermediate E
(Scheme 6) as a white amorphous solid. To a stirred solution of
derivative E in MeOH were added triethylamine and 5-ketohexanoic
anhydride.30 After 10 min, the solution was concentrated under reduced
pressure. To the residue was added water, and the mixture was stirred
with a magnetic stirring bar. The flask containing the solution was
immersed in ice−water. The clear solution was siphoned off with a
Pasteur pipet, the tip of which was covered with tissue paper. The
solution so obtained was freeze-dried. To the residue was added 0.02 M
pyridine−acetate buffer. The mixture was filtered through a pad of tissue
paper to remove residual solid particles. The clear solution was passed
through a Biogel P-4 column using 0.02 M pyridine-acetate as the eluant.
The fractions containing carbohydrates were pooled and freeze-dried.
The residue was dissolved in water, and the solution was freeze-dried.
This cycle was repeated two more times to afford compound F as a white
amorphous solid.
with the lipopolysaccharides of either SD or of E. coli O148, in
both directions. The dot blot assays indicate the importance of
the length of the oligosaccharide chains. BSA conjugates of the
monosaccharides galactose and glucose failed to react with either
of the antisera, and the conjugate of the tetrasaccharide 1 reacted
weakly as compared with the conjugates of the hexa- to
dodecasaccharides 2−7, which reacted with comparable
intensities. These qualitative observations support the view
that a chain length exceeding a complete repeating unit is
necessary for specific antigenic recognition by either SD or E.coli
O148 antibodies. Further proof for the cross-reactivity was
provided by competitive inhibition of binding between sera
raised by the neoglycoconjugates and their respective homolo-
gous LPS. The observed cross-reactivity suggests that anti-O-SP-
based immunity against one of these organisms would provide
protection to both, an observation that should be conducive to a
single monovalent vaccine against the two organisms.
EXPERIMENTAL SECTION
■
General Chemical Synthesis. All chemicals were commercial
grade and used without purification. Solvents for chromatography were
distilled prior to use. All glycosylation reactions were carried out after
drying the reacting partners at 10 μm or less for 12 h except for the
reaction with imidate 11 that was used immediately after its preparation.
Glycosylation reactions as well as preparation of trichloroacetimidates
were carried out under argon. Column chromatography was performed
on silica gel 60 (0.040−0.063 mm), and thin layer chromatography was
performed on glass-supported silica gel layers or on HPTLC plates.
Visualization was carried out by inspection under UV light (254 nm), by
iodine adsorption, and by charring using a solution of ammonium
cerium(IV) sulfate and ammonium molybdate in sulfuric acid. Column
chromatography was performed on silica gel 60 (230−400 mesh).
Routine 1H and 13C NMR spectra were recorded at 500 and 125 MHz,
respectively, using CDCl3 as the solvent unless indicated otherwise.
Chemical shifts are recorded in ppm relative to internal references. For
1H: 0.00 for (CH3)4Si, 3.30 for CD2HOD, or 2.225 for acetone. For 13C:
48.90 for CD3OD, 31.00 for acetone, or 77.00 for CDCl3. Glycoside 41
was examined as its solution in D2O, but for 42 and 14, the D2O lock
signals were found to be insensitive to shimming. Therefore, spectra for
41 and 42 were recorded for solutions in D2O with 20% v/v of acetone-
d6 present. The 1H and 13C NMR spectra of oligosaccharides 1−7 were
measured in D2O. The spectra of 2, 3, 5, and 6 for which partial
assignments are presented were measured in D2O/acetone-d6 (4:1 v/v).
Interchangeable assignments are denoted with an asterisk. The
monosaccharide residues are denoted with subscripted capital letters
starting with A for the reducing end unit. Because of the instability of the
acetone-d6 lock signal in the presence of a strong D2O resonance, the
best procedure found was to shim on acetone-d6, then lock on D2O for
data acquisition. The methods used for 1D and 2D NMR data
acquisition were similar to those reported by us recently for a set of
glycolipids.41 Atmospheric pressure electrospray ionization mass
spectrometry (API-ES-MS) was carried out on an LC/MSD SL
spectrometer.
Synthesis and/or Characterization of the New Compounds
Described in This Paper. 5-{2-[(5′-Oxohexanoyl)amino]ethyl-
amino}carbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-glucopyra-
nosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-
rhamnopyranoside (1). 1H NMR (D2O, partial) δ 5.53 (d, 1H, J = 3.6
Hz), 5.11 (br s, 1H), 4.98 (d, 1H, J = 3.6 Hz), 4.80 (br s, 1H), 4.13 (dd,
1H, J = 3.6 Hz, J = 10.4 Hz), 3.30 (br s, 4H), 2.58 (t, 2H, J = 7.4 Hz),
2.25−2.24 (m, 4H), 2.20, 2.04 (2 s, 2 × 3H), 1.83−1.77 (m, 2H), 1.64−
1.54 (m, 4H), 1.40−1.37 (m, 2H), 1.31, 1.29 (2 d, 2 × 3H); 13C NMR
(D2O) δ 216.2, 177.9, 177.0, 174.8, 102.2, 100.2, 98.5, 94.9, 77.1, 75.89,
75.85, 73.1, 72.9, 72.6, 72.5, 71.7, 71.0, 70.7, 70.5, 70.0, 69.8, 69.4, 68.4,
67.4, 60.8, 60.7, 52.6, 42.8, 39.3, 39.2, 36.5, 35.6, 30.0, 28.9, 25.8, 25.7,
22.9, 20.33, 20.28, 17.4, 17.3. HRMS m/z calcd for [C40H69N3O22]H+:
944.4451. Found: 944.4449.
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl 2-
acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyrano-
syl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-
J
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The Journal of Organic Chemistry
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Table 7. 1JC‑1,H‑1 Coupling Constants (Hz) of the E. coli O148 Tetra- (41), Octa- (42), and Dodecasaccharide 14 5-
Methoxycarbonylpentyl Glycosides
tetrasaccharide residues
octasaccharide residues
A
B
C
D
A
B
C
D
E
F
G
H
Rha
170.0
GlcNAc
171.6
Glc
Rha
Rha
170.0
GlcNAc
171.0
Glc
Rha
Rha
170.6
GlcNAc
172.0
Glc
Rha
1JC‑1,H‑1
175.8
172.6
174.4
173.0
174.4
172.4
dodecasaccharide residues
A
B
C
D
E
F
G
H
I
J
K
L
Rha
GlcNAc
168.9
Glc
Rha
Rha
171.1
GlcNAc
168.9
Glc
Rha
Rha
171.1
GlcNAc
168.9
Glc
Rha
172.8
1JC‑1,H‑1
169.9
175.3
171.8
175.3
171.8
175.3
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-D-
glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-
(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-
α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyrano-
syl)-(1→3)-α-L-rhamnopyranoside (3). 1H NMR (D2O, partial) δ 5.56
(d, 1H, J = 3.7 Hz, H-1C), 5.39 (d, 1H, J = 3.9 Hz, H-1G), 5.12 (d, 1H, J =
1.6 Hz, H-1E), 5.10 (d, 1H, J = 1.5 Hz, H-1D), 5.05 (d, 1H, J = 3.6 Hz, H-
1F), 4.98 (d, 1H, J = 3.7 Hz, H-1B), 4.80 (d, 1H, J = 1.7 Hz, H-1A), 4.22,
4.16 (2 br, 2 × 1H), 3.30 (br, 4H), 2.57 (t, 1H, J = 7.3 Hz), 2.24−2.20
(m, 4H), 2.19 (s, 3H), 2.03 (s, 6H), 1.80 (m, 2H), 1.65−1.54 (m, 4H),
1.39−1.26 (m, 11H); 13C NMR (D2O) δ 216.3, 178.0, 177.1, 174.9,
174.8, 102.72 (JC‑1,H‑1 = 175.1 Hz, C-1E), 102.15 (JC‑1,H‑1 = 174.3 Hz, C-
1D), 100.33 (JC‑1,H‑1 = 178.1 Hz, C-1G), 100.28 (JC‑1,H‑1 = 170.7 Hz, C-
1A), 98.49 (JC‑1,H‑1 = 177.3 Hz, C-1C), 95.11 (JC‑1,H‑1 = 172.9 Hz, C-1F),
94.95 (JC‑1,H‑1 = 173.3 Hz, C-1B), 78.9, 78.6, 77.4, 76.3, 76.0, 75.9, 73.6,
73.2, 73.02, 72.98, 72.5, 72.4, 72.1, 71.8, 71.2, 71.14, 71.08, 70.4, 70.2,
70.2, 69.9, 69.8, 69.5, 68.6, 67.6, 67.5, 60.93, 60.90, 60.83, 52.7, 43.0,
39.4, 39.3, 36.6, 35.7, 30.1, 29.0, 25.9, 25.8, 23.0, 20.4, 17.7, 17.54, 17.50.
HRMS m/z calcd for [C60H102N4O36]H+: 1455.6334. Found:
1455.6341.
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-L-
rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-
2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-
L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetami-
do-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (4).
1H NMR (D2O, partial) δ 5.53 (br, 2H), 5.10 (br, 2H), 5.08 (br, 1H),
5.02 and 4.97 (2 d, 2 × 1H, J ∼ 3.6 Hz), 4.79 (br, 1H), 4.22, 4.16, 4.13,
4.11 (4 br, 4 × 1H), 4.07 (br, 2H), 3.93 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz),
3.30 (m, 4H); 13C NMR (D2O) δ 216.3, 178.0, 177.1, 174.9, 174.8,
1s02.7, 102.3, 102.2, 100.2, 98.5, 95.0, 94.9, 78.8, 77.3, 77.2, 76.3, 75.93,
75.91, 75.7, 73.2, 73.1, 73.0, 72.6, 72.5, 74.5, 72.0, 71.8, 71.7, 71.1, 71.0,
70.8, 70.6, 70.3, 70.1, 70.0, 69.8, 69.4, 68.5, 67.5, 60.9, 60.7, 42.9, 39.4,
39.3, 36.5, 35.6, 30.0, 29.9, 25.9, 25.7, 22.9, 20.4, 17.6, 17.5, 17.4, 17.3.
HRMS m/z calcd for [C66H112N4O40]H+: 1601.6931. Found:
1601.7007.
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl 2-
acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyrano-
syl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-
2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyra-
nosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→
3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamno-
pyranoside (5). 1H NMR (D2O, partial) δ 5.561 (d, 1H, J = 3.6 Hz, H-
1C), 5.559 (d, 1H, J = 3.6 Hz, H-1G), 5.118 and 5.114 (2d, 2 × 1H, J ∼ 2.8
and 1.8 Hz, H-1E,I), 5.102 and 5.099 (2d, 2 × 1H, J ∼ 1.7 and 1.5 Hz, H-
1D,H), 5.06 (d, 1H, J = 3.8 Hz, H-1J), 5.04 (d, 1H, J = 3.6 Hz, H-1F), 4.98
(d, 1H, J = 3.7 Hz, H-1B), 4.80 (d, 1H, J = 1.7 Hz, H-1A), 4.98 (d, 1H, J =
3.7 Hz, H-1B), 4.80 (d, 1H, J = 1.7 Hz, H-1A), 4.23, 4.17 (2 br s, 2 × 1H),
4.17 (br s, 2H), 3.30 (s, 4H), 2.58 (t, 2H, J ∼ 7.1 Hz), 2.38 (t, J ∼ 7.0
Hz), 2.24, 2.22 (2 t, 2 × 1H. J ∼ 7.0 Hz), 2.20, 2.05, 2.04, 2.03 (4 s, 4 ×
3H), 1.83−1.77 (m, 2H), 1.65−1.55 (m, 4H), 1.34 (d, 6H, J ∼ 6.3 Hz),
1.31 (d, 3H, J = 6.3 Hz), 1.30 (d, 6H, J ∼ 6.3 Hz); 13C NMR (D2O) δ
216.2, 177.9, 177.0, 175.2, 174.80, 174.78, 102.74 (JC‑1,H‑1 = 172.2 Hz, C-
1E,I), 102.13 (JC‑1,H‑1 = 172.3 Hz, C-1D,H), 100.26 (JC‑1,H‑1 = 169.6 Hz, C-
1A), 98.49 (JC‑1,H‑1 = 175.3 Hz, C-1C,G), 95.01 (JC‑1,H‑1 = 170.8 Hz, C-1J),
94.93* (JC‑1,H‑1 = 170.9 Hz, C-1F), 94.88* (JC‑1,H‑1 = 174.1 Hz, C-1B),
78.8, 77.3, 76.26, 76.0, 75.9, 75.6, 73.1, 73.0, 72.6, 72.5, 72.0, 71.8, 71.7,
71.13, 71.08, 71.0, 70.5, 70.3, 70.1, 70.0, 69.96, 69.8, 69.4, 68.5, 67.6,
Figure 4. 1H-coupled 2D HSQC spectrum of (a) octasaccharide 42 and
(b) dodecasaccharide 14.
2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyra-
noside (2). 1H NMR (D2O, partial) δ 5.55 (d, 1H, J = 3.6 Hz, H-1c), 5.11
(d, 1H, J = 1.4 Hz, H-1E), 5.10 (d, 1H, J = 1.2 Hz, H-1D), 5.06 (d, 1H, J =
3.7 Hz, H-1F), 4.98 (d, 1H, J = 3.7 Hz, H-1B), 4.80 (d, 1H, J = 1.5 Hz, H-
1A), 3.30 (br, 4H), 2.57 (t, 2H, J = 7.3 Hz), 2.23, 2.21 (2 t, 2 × 2H, J ∼ 7
Hz), 2.19, 2.04, 2.03 (3 s, 3 × 3H), 1.80 (m, 2H), 1.63−1.54 (m, 4H),
1.38−1.27 (m, 11H); 13C NMR (D2O) δ 216.2, 177.9, 177.0, 175.2,
174.8, 102.70 (JC‑1,H‑1 = 171.9 Hz, C-1E), 102.09 (JC‑1,H‑1 = 173.1 Hz, C-
1D), 100.23 (JC‑1,H‑1 = 171.3 Hz, C-1A), 98.45 (JC‑1,H‑1 = 177.8 Hz, C-1C),
94.98 (JC‑1,H‑1 = 172.9 Hz, C-1F), 94.90 (JC‑1,H‑1 = 173.6 Hz, C-1B), 78.8,
77.3, 76.3, 76.0, 75.9, 73.1, 73.0, 72.6, 72.5, 72.0, 71.8, 71.7, 71.1, 71.0,
70.5, 70.3, 70.1, 70.0, 69.8, 69.4, 68.5, 67.6, 67.5, 61.0, 60.9, 60.8, 54.4,
52.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.9, 22.7, 20.4,
17.6, 17.5, 17.4. HRMS m/z calcd for [C54H91N4O31]H+: 1315.5643.
Found: 1315.5658.
L
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

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Article
Scheme 7. Synthesis of Neoglycoproteins by Oxime-Conjugation between Carbohydrates and Proteins
2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-
L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetami-
do-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→
3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acet-
amido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside
(7). 1H NMR (D2O, partial) δ 5.57 (br s, 3H), 5.13 (br s, 2H), 5.06 (br d,
2H, J = 3.5 Hz), 5.00 (br s, 1H, J = 3.5 Hz), 4.83 (br s, 1H), 4.26, 4.19 (2
br s, 2 × 2H), 3.33 (br s, 4H), 2.16 (t, 2H, J = 7.3 Hz), 2.27, 2.26 (2 t, 2 ×
2H, J ∼ 7 Hz), 2.10−2.04 (m, 9H), 1.86−1.80 (m, 2H), 1.67−1.57 (m,
4H), 1.42−1.30 (m, 18H); 13C NMR (D2O) δ 216.2, 177.9, 177.1,
174.84, 174.81, 174.8, 102.72, 102.69, 102.3, 102.2, 102.1, 100.2, 98.5,
94.95, 94.92, 78.8, 77.3, 77.1, 76.3, 75.92, 75.90, 75.6, 73.20, 73.16,
73.02, 73.01, 72.96, 72.93, 72.92, 72.91, 72.6, 72.52, 72.48, 72.04, 72.02,
71.99, 71.8, 71.7, 71.10, 71.07, 71.06, 71.02, 70.99, 70.8, 70.6, 70.3, 70.2,
70.12, 70.08, 70.07, 70.06, 70.05, 70.0, 69.9, 69.85, 69.80, 69.4, 68.5,
67.5, 67.4, 60.93, 60.92, 60.91, 60.87, 60.86, 60.83, 60.76, 60.75, 60.73,
60.72, 60.70, 52.7, 42.9, 39.39, 39.37, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9,
25.7, 22.9, 20.4, 17.62, 17.59, 17.51, 17.47, 17.43, 17.40, 17.39, 17.37.
HRMS m/z calcd for [C92H155N5O58]H +: 2258.9411. Found:
2258.9670.
Table 8. Oligosaccharides Related to S. dysentariae Type 1
Lipopolysaccharide That Were Used in This Study
[α-L-Rhap-1,2-α-D-Galp-1,3-α-D-GlcpNAc-1,3-α-L-Rhap]2-R
43
44
α-D-GlcpNAc-1,3-α-L-Rhap-1,3-[α-L-Rhap−1,2-α-D-Galp-1,3-α-D-
GlcpNAc-1,3-α-L-Rhap]2-R
α-D-Galp-1,3-α-D-GlcpNAc-1,3-α-L-Rhap-1,3-[α-L-Rhap−1,2-α-D-Galp- 45
1,3-α-D-GlcpNAc-1,3-α-L-Rhap]2-R
[α-L-Rhap-1,2-α-D-Galp-1,3-α-D-GlcpNAc-1,3-α-L-Rhap]3-R
46
47
α-L-Rhap-1,3-[α-L-Rhap−1,2-α-D-Galp-1,3-α-D-GlcpNAc-1,3-α-L-
Rhap]3-R
67.5, 61.0, 60.9, 54.4, 52.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9,
25.7, 22.92, 22.91, 22.7, 20.4, 17.6, 17.5, 17.4. HRMS m/z calcd for
[C80H135N5O49]H+: 1950.8304. Found: 1950.8314.
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-D-
glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-
(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-
α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyrano-
syl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→
2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyra-
(2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-α-L-rhamnopyrano-
syl-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acet-
amido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-
benzoyl-4-O-benzyl-α-L-rhamnopyranose trichloroacetimidate (8).
To a solution of 30 (13.0 g, 8.7 mmol) in CH2Cl2 (50 mL) were added
CCl3CN (15 mL) and DBU (0.3 mL) under cooling in ice−water. The
mixture was allowed to reach room temperature. After 1 h the solution
was concentrated, and the residue purified by column chromatography
using hexanes−EtOAc 3:2 containing 0.1% Et3N to afford 8 (11.1 g,
1
nosyl)-(1→3)-α-L-rhamnopyranoside (6). H NMR (D2O, partial) δ
5.56 (br d, 2H, J ∼ 2.6 and 2.8 Hz, H-1C,G), 5.39 (d, 1H, J = 3.9 Hz, H-
1K), 5.12 (br s, 2H, H-1E,I), 5.10 (br s, 2H, H-1D,H), 5.05 (d, 1H, J = 3.8
Hz, H-1J), 5.04 (d, 1H, J ∼ 3.9 Hz, H-1F), 4.98 (d, 1H, J = 3.6 Hz, H-1B),
4.80 (d, 1H, J = 1.5 Hz, H-1A), 4.23, 4.17 (2 br s, 2 × 2H), 3.30 (s, 4H),
2.23, 2.22 (2 t, 2 × 1H, J ∼ 7 Hz), 2.19, 2.04, 2.03 (3 s, 3 × 3H), 1.83−
1.77 (m, 2H), 1.64−1.53 (m, 4H), 1.33 (d, 6H, J ∼ 6.3 Hz), 1.30 (d, 3H,
J = 6.3 Hz), 1.29 (d, 6H, J = 6.3 Hz); 13C NMR (D2O) δ 216.3, 178.0,
177.1, 174.9, 174.83, 174.81,102.69 (JC‑1,H‑1 = 170.7 Hz, C-1E,I), 102.13
(JC‑1,H‑1 = 172.7 Hz, C-1D,H), 100.32 (JC‑1,H‑1 = 172.5 Hz, C-1K), 100.26
(JC‑1,H‑1 = 172.1 Hz, C-1A), 98.48 (JC‑1,H‑1 = 174.9 Hz, C-1C,G), 95.09
(JC‑1,H‑1 = 172.5 Hz, C-1J), 94.93*(JC‑1,H‑1 = 172.4 Hz, C-1F),
94.88*(JC‑1,H‑1 = 172.4 Hz, C-1B), 78.8, 78.5, 77.3, 76.3, 75.9, 73.6,
73.2, 73.0, 72.9, 72.5, 72.4, 72.0, 71.8, 71.2, 71.1, 71.0, 70.3, 70.1, 70.0,
69.8, 69.7, 69.4, 68.5, 67.6, 67.5, 60.87, 60.86, 60.8, 52.7, 42.9, 39.4, 39.3,
36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.9, 20.4, 17.6, 17.5, 17.4. HRMS m/z
calcd for [C86H145N5O54]H +: 2112.8832. Found: 2112.8816.
1
78%) as an amorphous material: H NMR (CDCl3, partial) δ 8.75 (s,
1H), 8.15, 8.13, 8.00, 7.96 (4 s, 4 × 1H), 7.67−7.59 (m, 2H), 7.55−7.49
(m 2H), 7.37−7.02 (m, 24H), 6.29 (d, 1H, J = 11.7 Hz), 5.75 (d, 1H, J =
9.5 Hz), 5.63−5.60 (2 br s, 2 × 1 H), 5.40 (dd, 1H, J = 3.5 Hz, J = 9.2
Hz), 5.19 (t, 1H, J = 9.6 Hz), 5.16 (br s, 1H), 4.93 (d, 1H, J = 3.4 Hz
each), 4.79 (d, 1H, J = 3.2 Hz), 4.74, 4.69, 4.66, 4.62, 5.53 (5 d, 5 × 1H, J
∼ 11 Hz), 4.52 (d, 2H, J ∼ 11 Hz), 4.45 (dt, 1H, J = 3.4 Hz, J = 10.0 Hz),
4.35, 4.34, 4.30 (3 d, 3 × 1H, J ∼ 11 Hz), 4.24 (dd, 1H, J = 3.3 Hz, J =
10.0 Hz), 3.49 (t, 1H, J = 9.6 Hz), 2.13, 2.00, 1.79 (3 s, 3 × 3H), 1.40,
1.09 (2 d, 2 × 3H, J = 6.3 each); 13C NMR (CDCl3) δ 170.6, 170.4,
169.4, 165.9, 165.5, 165.2, 160.1, 138.2, 137.9, 137.7, 137.6, 137.1, 134.2,
133.4, 129.84, 129.80, 129.77, 129.2, 128.9, 128.7, 128.44, 128.39, 128.3,
128.35, 128.3, 128.25, 128.18, 128.1, 128.05, 128.02, 127.98, 127.94,
127.8, 127.7, 127.54, 127.48, 127.35, 127.32, 99.6, 99.4, 96.8, 94.8, 90.6,
80.9, 78.8, 78.42, 78.38, 78.0, 77.6, 75.7, 75.44, 75.37, 74.8, 74.4, 73.9,
5-{2-[(5′-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-L-
rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-
M
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Table 9. Composition of Protein Conjugates of Synthetic Oligosaccharides Related to S. dysenteriae Type 1 and E. coli O148, and
a b c d
, , ,
the Geometric Means of Their Anti-LPS Serum IgG
item
no.
conjugate/
OS size
nonreducing
terminus
average molecular mass of
conjugate [kDa]
average number of OS
chains per protein
protein sugar
ratio (w/w)
anti-SD LPS IgG
anti-Ec148 LPS IgG
e
e
GM[EU]
GM [EU]
1
1-EC-BSA/4-
Rha(1)
GlcNAc
Glc
92
20
18
15
14
15
12
11
13
12
7
4.9
3.6
3.7
3.6
2.6
3.0
2.9
2.6
2.8
6.6
2.9
2.9
4.1
3.9
ND
ND
ND
33.3
4.6
ND
ND
ND
155.6
14.7
ND
39.8
56.4
45.7
ND
33.7
48.0
ND
ND
mer
2
2-EC-BSA/
6mer
95
3
3-EC-BSA/
7mer
94
4
4-EC-BSA/8-
mer
Rha(2)
GlcNAc
Glc
95
5
5-EC-BSA/
10-mer
102
98
6
6-EC-BSA/
11mer
ND
12.9
11.2
14.5
14.4
11.8
9.5
7
6-EC-DT/
11mer
Glc
86
8
7-EC-BSA/
12mer
Rha(2)
Rha(2)
Rha(2)
GlcNAc
Gal
102
90
9
7-EC-DT/
12mer
10
11
12
13
14
43-SD-DT/
8mer
74
44-SD-DT/
10mer
86
12
11
7
45-SD-DT/
11mer
86
46-SD-DT/
12mer
Rha(2)
Rha(1)
79
17.9
13.5
47-SD-DT/
13mer
80
7
a
The immune responses to the BSA conjugates of oligosaccharides 1, 2, 3, and 6 were not investigated. These conjugates were tested for their
b
antigenicity as reported in the Immunoblotting section. Molecular weights: BSA, 66.5 kDa; rDT, 58.3 kDa; aminooxy-derivatized BSA, 73 kDa;
c
d
aminooxy-derivatized rDT, 63 kD. Average numbers of aminooxy groups in the derivatized BSA: 28; in the derivatized rDT: 20. Abbreviations:
BSA, bovine serum albumin; DT, recombinant diphtheria toxin; EC, Escherichia coli; EU, elisa unit; GM, geometric mean; IgG, immunoglobulin G;
LPS, lipopolysaccharide; ND, not determined; Rha(1), the rhamnose unit to which GlcNAc is attached; Rha(2), the rhamnose unit which is linked
e
to either Gal or Glc; OS, oligosaccharide; SD, Shigella dysenteriae type 1. All groups vs control: p < 0.001.
73.5, 71.5, 71.4, 70.1, 68.7, 68.6, 68.3, 68.0, 67.8, 60.9, 51.9, 40.4, 23.1,
21.0, 20.7, 18.2, 17.4.
mL) was added Cl3CCN (25 mL, 243 mmol) under ice-cooling. After 30
min at room temperature, the mixture was extracted with brine 3 times
followed by drying (Na2SO4). Removal of the volatiles under reduced
pressure afforded a syrup, which was used in the next step without
further purification.
2-(Trimethylsilyl)ethyl (3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-
(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-
(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (10). A mix-
ture of 24 (approximately 35 g, 27.0 mmol) in CH3CN (350 mL) and
water (60 mL) was treated with ammonium cerium(IV)nitrate (34 g, 62
mmol) in portions. The mixture was cooled to 0 °C and then was
decolorized with 10% aq NaHSO3. The solution was then extracted with
saturated aq NaHCO3. The mixture was filtered through a Celite layer,
and the solids were washed with CHCl3. The combined organic layers
were washed with brine several times. Concentration of the organic layer
yielded a syrupy residue, which was chromatograped through silica gel
using hexanes−EtOAc 5:1 → 2:1 as the eluant to afford 10 (17.8 g, 15
mmol) as a syrupy material: 1H NMR (CDCl3, partial) δ 8.12−8.05 (m,
2H), 7.58−7.53 (m, 1H), 7.43−7.41 (m, 2H), 7.38−7.19 (m, 16H),
7.19−7.15 (m, 2H), 7.10−7.07 (m, 2H), 5.46 (m, 1H), 5.14 (t, 1H, J =
9.9 Hz), 5.11 (d, 1H, J = 3.6 Hz), 4.87 (d, 1H, J = 3.6 Hz), 4.86 (d, 1H, J
= 11.0 Hz), 4.84 (d, 1H, J = 12 Hz), 4.81 (d, 1H, J = 11.8 Hz), 4.76 (d,
1H, J = 11.5 Hz), 4.73 (d, 1H, J = 11.6 Hz), 4.68 (d, 1H, J = 11.0 Hz),
4.30 (d, 1H, J = 12.0 Hz), 4.20 (d, 1H, J = 12.0 Hz), 3.36 (dd, 1H, J = 6.2
Hz, J = 10.0 Hz), 3.29 (dd, 1H, J = 8.6 Hz, J = 10.0 Hz), 2.04, 1.90, 1.61
(3 s, 3 × 3H), 1.44 (d, 3H, J = 6.3 hz), 1.00−0.88 (m, 2H), 0.03 (s, 9H);
13C NMR (CDCl3) δ 170.6, 170.1, 169.8, 165.2, 138.4, 138.0, 137.7,
5-(Methoxycarbonyl)pentyl (2-O-benzoyl-4-O-benzyl-3-O-chlor-
oacetyl-α-L-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-D-gluco-
pyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-gluco-
pyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside
(13). To a stirred solution of compounds 8 (3.0 g, 1.83 mmol) and 31
(375 mg, 2.5 mmol) in CH2Cl2 (35 mL) was added TMSOTf (15 μL)
under external ice−water cooling. After 1 h the solution was treated with
Et3N (∼0.1 mL) followed by removal of the volatiles under vacuum.
Column chromatography of the residue using hexanes−EtOAc 2:1 →
3:2 as the eluant gave 13 (2.4 g, 81%) as an amorphous substance: 1H
NMR (CDCl3, partial) δ 8.13−8.11 (m, 2H), 7.98−7.97 (m, 2H), 7.66−
7.59 (m, 2H), 7.52−7.45 (m, 4H), 7.35−7.15 (m, 19H), 7.09−7.02 (m,
6H), 5.72 (d, 1H, J = 5.7 Hz), 5.60 (dd, 1H, J = 1.7 Hz, J = 3.5 Hz), 5.40
(dd, 1H, J = 3.6 Hz, J = 10.3 Hz), 5.39 (br s, 1H), 5.19 (t, 1H, J = 9.9 Hz),
5.14 (br d, 1H, J ∼ 1.8 Hz), 4.90 (d, 1H, J = 3.5 Hz), 4.80 (d, 1H J = 1.9
Hz), 4.78 (d, 1H, J = 3.3 Hz), 4.74 (d, 1H, J = 10.5 Hz), 4.43 (dt, 1H, J =
3.5 Hz, J = 10.2 Hz), 4.17 (dd, 1H, J = 2.9 Hz, J = 9.7 Hz), 3.67 (s, 3H),
3.37 (t, 1H, J = 9.6 Hz), 2.34 (t, 1H, J = 7.6 Hz), 2.14, 2.01,1.75 (3 s, 3 ×
3H), 1.69−1.55 (m, 4H), 1.42−1.37 (m, 2H), 1.36, 1.08 (2 d, 2 × 3H, J
= 6.3 Hz each); 13C NMR (CDCl3) δ 174.0, 170.7, 170.5, 169.4, 166.0,
165.7, 165.3, 138.3, 138.0, 137.8, 137.5, 137.4, 133.9, 133.4, 129.8−
127.3, 99.8, 99.5, 97.4, 96.5, 80.9, 79.5, 78.8, 78.4, 78.0, 77.9, 75.7, 75.4,
75.2, 75.1, 74.4, 73.9, 73.5, 71.4, 70.4, 70.1, 68.58, 68.55, 68.1, 98.0, 67.9,
67.7, 60.8, 51.9, 51.4, 40.4, 33.8, 29.0, 25.6, 24.6, 23.0, 21.1, 20.7, 18.2,
17.4. HRMS m/z calcd for [C88H100ClNO26]H+: 1622.6300. Found:
1622.6238.
137.3, 133.5, 129.7−126.9, 99.7, 96.9, 93.9, 82.8, 79.7, 77.1, 75.41, 75.39,
75.1, 74.3, 73.2, 72.9, 72.7, 70.6, 70.4, 68.8, 68.7, 68.0, 67.8, 65.4, 61.1,
51.4, 22.5, 20.7, 20.6, 18.0, 17.8, −1.5. HRMS m/z calcd for
[C64H79NO18Si]H+: 1178.5145. Found: 1178.5162.
3,4,6-Tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-D-glucopyranosyl
trichloroacetimidate (11). To a stirred mixture of compound 22 (12 g,
21 mmol) and Cs2CO3 (1.2 g, 3.7 mmol) in anhydrous CH2Cl2 (100
N
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Figure 5. Western immunoblot of S. dysenteriae type 1 and E. coli O148 LPS with sera induced by whole bacteria or synthetic oligosaccharides bound to
the recombinant diphtheria toxin (DT). The terminal sugars are as follows: 10mer, GlcNAc; 11mer, Gal; 12 and 13mers, Rha. Abbreviations: EC, E. coli
O148; SD, S. dysenteriae type 1.
3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-
glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-
Table 10. Competitive Inhibition of Anti-S. dysenteriae Type 1
Serum (Induced By rDT/Sd1-12mer) Binding to the
Homologous LPS by Different Dosages of S. dysenteriae Type
1, E. coli O148, or B. bronchiseptica RB50 O-SPs
1
(1→3)-α-L-rhamnopyranoside (14). For H and 13C NMR data, see
Tables 2, 4, 6, and 7.
1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranose (18). A
stirred solution of compound 17 (5.0 g) in Me3SiOAc (120 mL) was
refluxed for 2 h. The solution was concentrated, and the residue purified
by column chromatography using hexanes−EtOAc (2:1) to afford 18
(4.6 g, 93%) as a syrup: 1H NMR (CDCl3) δ 7.41−7.12 (m, 15H), 5.61
(d, 1H, J = 7.8 Hz), 5.11 (dd,1 H, J = 8.3 Hz, J = 9,4 Hz), 4.80, 4.77, 4.67,
4.62, 4.54, 4.49 (6 d, J ∼ 11 Hz each), 3.82 (t, 1H, J = 9.2 Hz), 3.77−3.69
(m, 3H), 3.61−3.57 (m, 1H), 2.07, 1.92 (2 s, 2 × 3H); 13C NMR
(CDCl3) δ 169.4, 138.0−127.7, 92.2, 82.7, 75.7, 75.1, 75.0, 73.5, 72.1,
inhibition (%)
inhibitor per well (μg)
inhibitor
0.04
0.2
5
80
S. dysenteriae type 1 O-SP
E. coli O148 O-SP
23
21
5
39
37
9
69
71
14
75
79
25
B. bronchiseptica O-SP
+
68.0, 20.9, 20.7. HRMS m/z calcd for [C31H34O8]NH4 : 552.2597.
Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-gluco-
pyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-
α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glu-
copyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→
Found: 552.2593.
Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-β-D-glucopyranoside
(19). To a stirred solution of diacetate 18 (2.4 g, 4.5 mmol), PhSSiMe3
(2.0 mL) in CH2Cl2 (15 mL) was added BF3·Et2O (0.8 mL) under
cooling with ice−water. The cooling bath was then removed, and the
solution was allowed to reach room temperature. After 90 min total
reaction time, to the solution was added Et3N (2 mL) followed by
removal of the volatiles under reduced pressure to yield a solid, which
was stirred in MeOH for 14 h. Filtration followed by drying afforded
crystalline 19 (2.2 g, 84%): mp 114−116 °C; 1H NMR (CDCl3) δ 7.52−
7.48 (m, 2H), 7.33−7.19 (m, 18H), 5.02 (m, 2H), 4.80−4.77 (m, 2H),
4.67 (d, 1H, J = 11.0 Hz), 4.61 (d, 1H, J = 10.0 Hz), 4.59 (d, 1H, J = 12.0
Hz), 4.57 (d, 1H, J = 11.0 Hz), 4.53 (d, 1H, J = 12.0 Hz), 3.79 (dd, 1H, J
= 1.8 Hz, J = 11.0 Hz), 3.72 (dd, 1H, J = 4.8 Hz, J = 11.0 Hz), 3.70−3.65
(m, 2H), 3.54 (m, 1H), 1.99 (s, 3H); 13C NMR (CDCl3) δ 169.4, 138.1,
138.0, 137.8, 132.9, 132.2, 128.7−127.5, 85.9, 84.3, 79.3, 77.7, 75.2, 75.0,
Table 11. Competitive Inhibition of Anti-E. coli O148 Serum
(Induced By rDT/EcO148-12mer) Binding to the
Homologous LPS by Different Dosages of S. dysenteriae Type
1, E. coli O148, or B. bronchiseptica RB50 O-SPs
inhibition (%)
inhibitor per well (μg)
inhibitor
0.04
0.2
5
80
S. dysenteriae type 1 O-SP
E. coli O148 O-SP
23
30
8
54
42
9
58
72
8
80
83
24
+
73.3, 71.7, 68.8, 20.9. HRMS m/z calcd for [C35H36O6S]NH4 :
602.2576. Found: 602.2556.
B. bronchiseptica O-SP
O
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Phenyl 3,4,6-tri-O-benzyl-1-thio-β-D-glucopyranoside (20). To a
solution of 19 (38 g) in CH2Cl2 (150 mL) were added sequentially
MeOH (50 mL) and NaOMe (10 mL of a 25% solution of NaOMe in
MeOH) at room temperature. After 4 h the solution was treated with
Dowex50 (H+) until its pH dropped to approximately 3. The solids were
removed by filtration, and the solution so obtained was extracted with
5% aq NaHCO3 and then washed with H2O twice. The organic layers
were combined and concentrated. Hexanes were added to and
evaporated from the residue. The solids so obtained were triturated
with hexanes followed by filtration to afford 20 (34 g, 97%) as a
2-(Trimethylsilyl)ethyl [3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-
α,β-D-glucopyranosyl]-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranoside (24). A mixture consisting of 23 (33 g, 10.9 mmol),
Et3N (7.5 mL), EtOAc (30 mL), EtOH (140 mL), and 10% palladium-
on-charcoal (5.5 g) was stirred under hydrogen at 200 psi at room
temperature for 1 h. The mixture was treated with Ac2O (11 mL) and
then was filtered through a Celite layer followed by concentration. A
solution of the residue in CHCl3 was washed with water, dried
(Na2SO4), and concentrated. The material so obtained was used in the
1
next step without further purification: H NMR (CDCl3, partial) δ
1
crystalline solid: mp 74−76 °C; H NMR (CDCl3) δ 7.61−7.51 (m,
8.11−8.06 (m, 2H), 7.62−7.55 (m, 1H), 7.49−7.43 (m, 2H), 7.41−7.36
(m, 2H), 7.35−7.28 (m, 4H), 7.27−7.17 (m, 12H), 7.16−7.10 (m, 2H),
7.08−7.02 (m, 2H), 6.76−6.69 (m, 2H), 5.57 (d, 1H, J = 9.8 Hz), 5.42
(m, 1H), 5.19 (t, 1H, J = 9.8 Hz), 5.03 (d, 1H, J = 3.5 Hz), 3.71 (s, 3H),
2.02, 1.71, 1.52 (3 s, 3 × 3H), 1.41 (d, 1H, J = 6.3 Hz), 0.99−0−86 (m,
2H), 0.01 (s, 9H); 13C NMR (CDCl3) δ 170.8, 170.0, 169.2, 165.6,
159.3, 138.8, 138.5, 137.8, 137.7, 130.4−127.4, 113.8, 113.6, 99.2, 97.0,
95.2, 81.5, 80.1, 79.9, 77.5, 75.6, 75.4, 74.7, 73.9, 73.5, 72.9, 71.3, 69.5,
69.4, 68.1, 65.6, 55.3, 52.0, 22.9, 20.9, 20.8, 18.2, 18.0, −1.3. HRMS m/z
calcd for [C72H87NO19Si]H +: 1299.5720. Found: 1299.5760.
2H), 7.39−7.11 (m, 18H), 4.90, 4.83, 4.81, 4.60, 4.56, 4.53 (6 d, 6 × 1H,
J ∼ 11 Hz), 4.49 (d, 1H, J = 9.5 Hz), 3.78 (dd, 1H, J = 10.4 Hz), 3.72 (dd,
1H, J = 4.2 Hz, J = 10.4 Hz), 3.62−3.57 (m, 2H), 3.55−3.47 (m, 2H);
13C NMR (CDCl3) δ 138.4, 138.2, 138.0, 132.8, 131.8, 128.9−127.5,
88.0, 85.9, 79.3, 77.3, 75.2, 75.0, 73.3, 72.5, 68.9 . HRMS m/z calcd for
+
[C33H34O5S]NH4 : 560.2471. Found: 560.2458.
Phenyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-1-thio-β-D-glu-
copyranoside (21). To a stirred solution of 20 (33.5 g, 62 mmol) in
DMF (150 mL) was added NaH (5.0 g, ∼125 mmol of a 60% suspension
in mineral oil) in portions under ice−water cooling. After 15 min,
MBnCl (11 mL, 81 mmol) was added dropwise. The mixture was
allowed to reach room temperature in approximately 30 min. To the
stirred mixture was added MeOH (excess) under cooling with ice−
water. The volatiles were removed by distillation under reduced
pressure, and the residue was equilibrated between CHCl3 and water.
The organic layer was concentrated. Trituration of the residue in
hexanes followed by filtration afforded 21 (38.4 g, 91%) as a colorless
2-(Trimethylsilyl)ethyl (3,4,6-tri-O-benzyl-β-D-glucopyranosyl)-
(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-
(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (25). Com-
pound 25 (approximately 3.5 g, 3 mmol) was obtained after the isomer
1
10 was eluted as described above: H NMR (CDCl3, partial) δ 8.16−
8.09 (m, 2H), 7.69−7.63 (m, 1H), 7.58−7.50 (m, 1H), 7.39−7.17 (m,
27H), 7.16−7.13 (m, 2H), 7.12−7.06 (m, 2H), 5.81 (d, 1H, J = 8.9 Hz),
5.44 (br, 1H), 2.01, 1.91, 1.66 (3 s, 3 × 3H), 1.47 (d, 3H, J = 6.3 Hz),
1.00−0.88 (m, 3H), 0.03 (s, 9H); 13C NMR (CDCl3) δ 172.0, 170.7,
169.6, 166.8, 165.8, 138.8, 138.3, 138.2,137.9, 137.6, 137.5, 137.4, 134.0,
129.8−127.4, 104.1, 96.9, 94.7, 85.3, 84.3, 79.9, 79.7,78.6, 76.7, 76.6,
75.5, 75.3, 74.9, 74.8, 74.7, 74.5, 73.7, 73.6, 73.3, 73.2, 69.4, 69.0, 68.2,
68.09, 68.06, 67.6, 65.5, 61.4, 52.2, 42.5, 22.8, 20.7, 20.5,18.2, 17.9, −1.4.
API-ES-MS m/z calcd for [C64H79NO18Si]H+: 1178.5145. Found:
1178.5162.
1
crystalline solid: mp 86−87 °C; H NMR (CDCl3) δ 7.76−7.73 (m,
2H), 7.47−7.26 (m, 20H), 6.99−6.92 (m, 2H), 5.02, 4.95 (2 d, 2 × 1 H, J
= 11.3 Hz each), 4.94, 4.91 (2 br s, 2 × 1H), 4.77, 4.76 (2 d, 2 × 1H, J =
10.2 Hz), 4.70 (d, 1H, J = 12.0 Hz), 4.69 (d, 1H, J = 10.7 Hz), 4.63 (d,
1H, J = 12.0 Hz), 3.86 (s, 3H), 3.89−3.73 (m, 4H), 3.67−3.54 (m, 2H);
13C NMR (CDCl3) δ 159.3, 138.4, 138.2, 138.0, 133.8, 131.8, 130.1−
127.3, 113.8, 87.4, 86.7, 80.5, 79.0, 77.7, 75.7, 74.97, 74.95, 73.3, 68.9,
+
2-(Trimethylsilyl)ethyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-D-gluco-
pyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-gluco-
pyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside
(26). To a solution of compound 10 (4.3 g, 3.6 mmol) in CH2Cl2 (20
mL) were added C5H5N (5 mL), Ac2O (5 mL), and a catalytic amount
of 4-dimethylaminopyridine. The solution was kept at ∼45 °C for 1 h
followed by removal of the volatiles under reduced pressure. Toluene
was added to and removed from the residue several times to afford 26
(quant.) that was used in the next step without further purification: 1H
NMR (CDCl3, partial) δ 8.11−8.08 (m, 2H), 7.59−7.56 (m, 1H), 7.45−
7.42 (m, 2H), 7.36−7.18 (m, 16H), 7.15−7.14 (m. 2H), 7.11−7.07 (m,
2H), 5.89 (d, 1H, J = 9.5 Hz), 5.47 (m, 1H), 5.14 (t, 1H, J = 9.7 Hz),
5.12, 5.08 (2d, 2 × 1H, J ∼ 3.6 Hz), 4.82 (d, 1H, J = 1.8 Hz), 4.76 (dd,
1H, J = 7.4 Hz, J = 11.0 Hz), 4.61 (d, 1H, J = 11.5 Hz), 4.45 (dd, 1H, J =
3.5 Hz, J = 10.3 Hz), 4.28−4.25 (m 2H), 4.02 (dd, 1H, J = 9.3 Hz, J =
10.8 Hz), 3.54−3.46 (m, 3H), 3.34 (dd, 1H, J = 6.8 Hz, J = 9.5 Hz), 3.30
(dd, 1H, J = 9.5 Hz, J = 10.0 Hz), 2.04, 1.97, 1.81, 1.60 (4 s, 4 × 3H), 1.42
(d, 3H, J = 6.3 Hz), 1.00−0.88 (m, 2H), 0.02 (s, 9H); 13C NMR
(CDCl3) δ 171.0, 170.6, 169.9, 169.2, 165.2, 138.3, 138.0, 137.7, 137.4,
133.6, 129.7−126.9, 97.0, 95.5, 93.6, 79.9, 79.8, 77.5, 75.5, 76.2, 74.4,
73.3, 73.0, 72.8, 72.7, 70.8, 70.3, 68.8, 68.7, 68.0, 67.7, 65.4, 61,2, 51.1,
22.6, 20.75, 20.68, 20.64, 18.0, 17.8, −1.5. HRMS m/z calcd for
[C66H81NO19Si]H+: 1220.5250. Found: 1220.5238.
55.2. HRMS m/z calcd for [C41H42O6S]NH4 : 680.3046. Found:
680.3057.
3,4,6-Tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-D-glucopyranose
(22). To a stirred mixture of 21 (33.0 g, 48 mmol), CH2Cl2 (500 mL),
and water (2 mL) was added (CF3CO2)2Hg (33.0 g, 77 mmol) under
cooling in ice−water. After 5 min the mixture was treated with saturated
aqueous KI (∼50 mL). The organic layer was separated and washed with
water. The residue obtained after removal of the volatiles was purified by
column chromatography using hexanes−EtOAc 2:1 → 3:2 as the eluant
1
to afford 22 (27.7 g, 97%) as a colorless syrup: H NMR (CDCl3,
partial) δ 7.36−7.22 (m, 15H), 7.16−7.09 (m, 2H), 6.85−6.80 (m, 2H),
5.15 (d, J = 3.6 Hz), 4.93, 4.82, 4.81, 4.69, 4.61, 4.58 (6 d, 6 × 1H, J ∼ 11
Hz), 3.94 (t, J = 9.0 Hz), 3.78 (s, 3H); 13C NMR (CDCl3, partial) δ
159.4, 138.7, 138.2, 137.8, 129.9−127.6, 113.9, 91.3, 81.7, 79.6, 77.6,
75.6, 75.0, 73.4, 72.8, 70.2, 68.6, 55.2. HRMS m/z calcd for
+
[C35H38O7]NH4 : 588.2961. Found: 588.2961.
2-(Trimethylsilyl)ethyl [3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-
α,β-D-glucopyranosyl]-(1→3)-(4,6-di-O-acetyl-2-azido-2-deoxy-α-D-
glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyra-
noside (23). To a stirred solution of 11 prepared from hemiacetal 22
(15.0 g, 26.3 mmol) and 12 (12.0 g, 16.4 mmol) in CH2Cl2 (100 mL)
was added TMSOTf (50 μL) at approximately −40 °C, and then the
solution was allowed to reach 0 °C in 2 h. The solution was extracted
with aq 5% NaHCO3, dried, and concentrated. Column chromato-
graphic purification of the residue using hexanes−EtOAc 10:1 → 2:1 as
(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-
acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-
O-benzoyl-4-O-benzyl-L-rhamnopyranose (27). A solution of 26 (4.5
g, 4.1 mmol) in a mixture of CH2Cl2 (20 mL) and CF3CO2H (100 mL)
was allowed to stand at rt for 4 h. Removal of the volatiles under reduced
pressure followed by column chromatography (hexanes−EtOAc 2:1 →
1:1) of the resulting syrup afforded 27 (3.3 g, 80%) as an amorphous
1
the eluant afforded 23 (19.0 g, 90%) as a colorless syrup: H NMR
(CDCl3, partial) δ 8.17−8.12 (m, 4H), 7.60−7.07 (m, 25H), 6.80−6.70
(m, 4H), 5.56 (m), 5.35 (d, J = 3.6 Hz), 5.15 (t, J = 10.0 Hz), 5.00 (d, J =
3.6 Hz), 3.70 (s, 3H), 2.06, 1.76 (2 s), 1.40 (d, J = 6.3 Hz), 1.02−0.88
(m), 0.03 (s, 9H); 13C NMR (CDCl3) δ 170.7, 169.3, 165.9, 159.4,
138.8−127.6, 98.6, 97.0, 93.0, 81.4, 79.5, 79.0, 77.3, 75.7, 75.4, 74.6,
73.3, 72.9, 72.7, 71.3, 69.3, 67.7, 67.4, 67.3, 65.2, 62.4, 55.0, 20.6, 17.9,
1
material: H NMR (D2O) δ 8.05−8.01 (m, 2H), 7.54−7.50 (m, 1H),
7.39−7.17 (m, 18H), 7.13−7.06 (m, 4H), 4.01 (dd, 1H, J = 9.0 Hz, J =
10.6 Hz), 3.83 (t, 1H, J ∼ 10.3 Hz), 3.80 (d, 1H, J = 3.3 Hz), 3.49, 3.45 (2
d, 2 × 1H, J ∼ 9.6 Hz), 2.02, 1.95, 11.78, 1.65 (4 s, 4 × 3H), 1.41 (d, 3H, J
= 6.3 Hz); 13C NMR (CDCl3) δ 171.1, 170.7, 170.6, 169.3, 165.2, 138.2,
+
17.8, −1.5. HRMS m/z calcd for [C70H83N3O18Si]NH4 : 1299.5785.
Found: 1299.5830.
P
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
137.9, 137.7, 137.2, 133.5−126.9, 95.5, 93.4, 92.1, 80.0, 79.8, 77.5, 75.6,
75.1, 74.4, 73.2, 72.9, 72.7, 72.3, 70.9, 70.3, 69.3, 68.7, 68.0, 67.7, 61.3,
51.3, 22.6, 20.72, 20.67, 18.1. HRMS m/z calcd for [C61H69NO19]H+:
1120.4542. Found: 1120.4515.
(CDCl3) δ 170.9, 170.7, 169.4, 166.0, 165.7, 165.3, 138.3, 137.9, 137.8,
137.6, 133.8, 133.4, 129.9−127.4, 99.7, 99.2, 95.9, 92.1, 80.9, 79.5, 78.5,
78.4, 78.0, 75.7, 75.3, 75.1, 74.6, 74.5, 73.9, 73.5, 71.4, 70.7, 70.1, 68.6,
68.5, 68.0, 67.8, 60.9, 51.8, 40.4, 23.0, 21.0, 20.7, 18.30, 18.27, 17.4.
HRMS m/z calcd for [C81H88ClNO24]H+: 1494.5463. Found:
1494.5516.
5-(Methoxycarbonyl)pentyl (2-O-benzoyl-4-O-benzyl-α-L-rham-
nopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-
(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-
(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (32). A mixture
consisting of compound 13 (3.3 g, 2.0 mmol), thiourea (3 g, 39.5
mmol), C5H5N (4 mL), and DMF (35 mL) was stirred at room
temperature for 12 h. The volatiles were removed by distillation under
reduced pressure. The residue was stirred in CHCl3 (50 mL) followed
by removal of the solids by filtration. Concentration afforded a syrup
that was purified by column chromatography using hexanes−EtOAc 2:1
→ 3:2 as the eluant to afford 32 (2.6 g, 83%) as an amorphous substance:
1H NMR (CDCl3, partial) δ 8.16, 8.00 (2 m, 2 × 2H), 7.68−7.59 (m,
2H), 7.55−7.45 (m, 4H), 7.41−7.18 (m, 1pH), 7.13−7.03 (m, 6H), 5.69
(d, 1H, J = 9.9 Hz), 5.42 (br s, 2H), 5.24 (br s, 1H), 5.19 (t, 1H, J = 9.8
Hz), 4.96 (d, 1H, J = 3.6 Hz), 4.77, 4.73, 4.69, 4.64, 4.56, 4.55 (6 d, 6 ×
1H, J ∼ 11 Hz each), 4.44 (dt, 1H, J = 3.4 Hz, J = 10.0 Hz), 3.69 (s, 3H),
2.36 (t, 2H, J = 7.6) 2.05, 2.03, 1.70 (3 s, 3 × 3H), 1.69−1.59 (m, 4H),
1.46−1.39 (m, 2H), 1.38, 1.28 (2 d, 2 × 3 H, J = 6.3 Hz each); 13C NMR
(CDCl3) δ 174.1, 170.8, 170.5, 169.0, 166.2, 165.7, 138.333, 138.327,
138.2, 137.74, 137.67, 134.0, 133.4, 129.9−127.4, 99.4, 98.5, 97.5, 95.7,
81.5, 81.4, 79.6, 78.0, 75.9, 75.7, 75.4, 75.3, 74.5, 74.4, 73.6, 73.3, 71.5,
70.6, 70.0, 69.3, 68.5, 68.4, 68.1, 68.0, 61.0, 51.9, 51.5, 33.9, 29.1, 25.7,
24.6, 23.0, 21.1, 20.8, 18.1, 17.9. HRMS m/z calcd for [C86H99NO25]H+:
1546.6584. Found: 1546.6604.
(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-
acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-2-
O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl trichloroacetimidate
(28). To a stirred solution of compound 27 (3.0 g, 2.7 mmol) in
anhydrous CH2Cl2 (50 mL) were added CCl3CN (3 mL) and DBU 0.9
mL) cooled at 0 °C. The solution was allowed to reach room
temperature in approximately 1 h. the solution was concentrated, and
the residue chromatographed through silica gel using hexanes−EtOAc
3:2 as the eluant, containing 0.1% Et3N to afford 28 (3.2 g, 95%) as an
amorphous material: 1H NMR (D2O) δ 8.70 (s, 1H), 8.10 (d, 2H, J = 7.8
Hz), 7.60 (t, 1H, J = 7.3 Hz), 7.50 (t, 2H, J = 7.9 Hz), 7.36−7.18 (m,
16H), 7.13, 7.09 (m, 2 × 2H), 6.27 (d, 1H, J = 1.7 Hz), 5.95 (d, 1H, J =
10.0 Hz), 5.71 (m, 1H), 5.16−5.10 (m, 3H), 4.78, 4.25 (2 × 1H, J = 11.5
Hz), 4.71−4.66 (m, 3H), 4.59 (d, 1H, J = 11.5 Hz), 4.45 (dt, 1H, J = 3.8
Hz, J = 10.6 Hz), 4.39 (d, 1H, J = 11.5 Hz), 4.32 (dd, 1H, J = 3.2 Hz, J =
9.8 Hz), 4.32 (d, 1H, J = 12.5 Hz), 4.22 (d, 1H, J = 12.5 Hz), 4.08−3.98
(m, 3H), 3.91 (m, 1H), 3.84 (dd, 1H, J = 9.0 Hz, J = 10.0 Hz), 3.81 (d,
2H, J = 3.1 Hz), 3.57 (t, 1H, J = 9.6 Hz), 3.48 (dd, 1H, J = 1.5 Hz, J = 10.2
Hz), 3.28 (t, 1H, J = 9.5 Hz), 3.21 (dd, 1H, J = 7.0 Hz, J = 10.1 Hz), 2.03,
1.97, 1.82, 1.64, (4 s, 4 × 3H), 1.46 (d, 3H, J = 6.3 Hz); 13C NMR
(CDCl3) δ 171.1, 170.6, 170.0, 169.3, 165.0, 164.9, 160.0, 138.3, 138.0,
137.4, 137.3, 133.9, 138.3, 138.0, 137.4, 137.3, 133.9, 129.9, 128.9−
127.4, 95.6, 95.0, 94.0, 90.6, 80,0, 79.1, 77.6, 75.6, 75.5, 74.5, 73.3, 73.0,
72.7, 72.5, 71.1, 70.8, 70.3, 68.9, 68.1, 66.9, 61.3, 51.1, 22.7, 20.8, 20.7,
18.1, 14.2.
2-(Trimethylsilyl)ethyl (2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-
α-L-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyrano-
syl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyrano-
syl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (29).
To a stirred solution of compounds 9 (36 g, 62.1 mmol) and 10 (16.4
g, 13.9 mmol) in CH2Cl2 (180 mL) was added TMSOTf (60 μL) under
cooling in ice−water. The solution was allowed to reach room
temperature. After 3 h the solution was treated with Et3N (0.5 mL).
The solution was washed sequentially with 2% aq NaHCO3 and water.
The solution was concentrated, and the residue purified by column
chromatography using hexanes−EtOAc 5:1 → 2:1 as the eluant to afford
29 (20.0 g, 91%) as an amorphous solid: 1H NMR (CDCl3, partial) δ
8.09 (m, 2H), 7.94 (m, 2H), 7.62−7.56 (m, 2H), 7.48−7.41 (m, 4H),
7.32−6.99 (m, 25H), 5.69 (d, 1H, J = 9.2 Hz), 5.57 (dd, 1H, J = 1.7 Hz, J
= 3.4 Hz), 5.37 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz), 5.35 (m, 1H), 5.16 (dd,
1H, J = 9.2 Hz, J = 10.3 Hz), 5.12 (d, 1H, J = 1.6 Hz), 4.86 (d, 1H, J = 3.4
Hz), 4.81 (d, 1H, J = 1.9 Hz), 4.76 (d, 1H, J = 3.5 Hz), 4.71 (d, 1H, J =
10.8 Hz), 4.64 (d, 1H, J = 11.3 Hz), 4.63 (d, 1H, J = 11.0 Hz), 4.59 (d,
1H, J = 11.6 Hz), 4.51 (d, 1H, J = 11.3 Hz), 4.47 (d, 1H, J = 11.3 Hz),
4.39 (dt, 1H, J = 3.3 Hz, J = 10.5 Hz), 4.18 (dd, 1H, J = 3.3 Hz, J = 10.5
Hz), 4.00 (m, 1H), 3.35 (t, 1H, J = 9.8 Hz), 2.12, 2.01, 1.74 (3 s, 3 × 3H),
1.37 (d, 3H, J = 6.3 Hz), 1.09 (d, 1H, J = 6.3 Hz), 1.03−0.89 (m, 2H),
0.03 (s, 9H); 13C NMR (CDCl3) δ 170.7, 170.4, 169.4, 165.9, 165.7,
165.3, 139.8−137.3, 133.9, 133.4, 99.8, 99.4, 96.8, 96.3, 80.9, 79.6, 78.7,
78.0, 77.8, 77.3, 75.7, 75.3, 75.2, 75.0, 74.4, 73.9, 73.5, 71.4, 70.5, 70.2,
68.7, 68.5, 68.1, 68.0, 67.8, 65.4, 60.9, 51.9, 40.4, 23.0, 21.1, 20.7, 18.2,
17.9, 17.4, −1.4. HRMS m/z calcd for [C86H100ClNO24Si]H+:
1594.6171. Found: 1594.6184.
(2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-α-L-rhamnopyrano-
syl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acet-
amido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-
benzoyl-4-O-benzyl-α-L-rhamnopyranose (30). To a solution of
compound 29 (21.5 g, 13.4 mmol) in CH2Cl2 (100 mL) was added
trifluoroacetic acid (400 mL) at room temperature. After 3 h the
volatiles were removed under reduced pressure. Toluene was added to
and evaporated from the residue four times followed by column
chromatographic purification using hexanes−EtOAc 1:1 → 2:3 as the
eluant to afford 30 (13.0 g, 65%) as a solid material: 1H NMR (CDCl3,
partial) δ 8.11−7.92 (m, 4H), 7.61 (t, 2H, J = 7.2 Hz), 7.46 (t, 4H, J = 7.7
Hz), 7.35−7.01 (m, 25H), 5.81 (d, 1H, J = 9.8 Hz), 5.59, 5.46 (2 br, 2 ×
1H), 5.39 (dd, 1H, J = 3.6 Hz, 9.5 Hz), 5.24 (br, 1H), 5.15 (m, 2H), 2.11,
2.01, 1.76 (3 s, 3 × 3H), 1.35, 1.09 (2 d, 2 × 3H, J = 6.3 Hz); 13C NMR
5-(Methoxycarbonyl)pentyl (2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamno-
pyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-
(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-
(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (34). To a stirred
solution of imidate 3329 (820 mg, 0.98 mmol) and compound 32 (650
mg, 0.42 mmol) in CH2Cl2 (10 mL) was added TMSOTf (8 μL) under
external cooling with ice−water. After 45 min the cooling bath was
removed, and the solution was treated with Et3N (∼0.1 mL).
Concentration under reduced pressure followed by column chromato-
graphic purification of the residue using hexanes−EtOAc 3:2 → 2:3 as
1
the eluant afforded 34 (0.85 g, 91%) as an amorphous substance: H
NMR (CDCl3, partial) δ 8.12−8.07 (m, 2H), 8.03−7.99 (m, 4H), 7.67−
7.57 (m, 2H), 7.53−7.44 (m, 6H), 7.37−7.10 (m, 29H), 7.04−6.97 (m,
2H), 5.75 (d, 1H, J = 9.7 Hz), 5.57 (dd, 1H, J = 1.5 Hz, J = 3.4 Hz), 5.41,
5.38 (2 br m, 2 × 1H), 5.36 (d, 1H, J = 9.7 Hz), 5.21, 5.20 (2 d, 2 × 1H, J
= 1.5 Hz each), 5.06, 4.98 (2 d, 2 × 1H, J = 9.7 Hz each), 5.02 (d, 1H, J =
7 Hz), 4.91 (d, 1H, J = 3.4 Hz), 4.15, 4.10 (2 dd, 2 × 1 H, J = 3.0, J = 9.9
Hz each), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.4 Hz), 1.98, 1.96, 1.91, 1.87,
1.77, 1.69, 1.45 (7 s, 7 × 3H), 1.66−1.57 (m, 4H), 1.43−1.40 (m, 2H),
1.37, 1.24, 1.11 (3 d, 3 × 3H, J = 6.3 Hz each); 13C NMR (CDCl3) δ
174.0, 171.1, 170.6, 170.5, 170.3, 169.9, 169.0, 168.9, 165.6, 165.3, 165.0,
138.3, 138.1, 137.7, 137.6, 137.3, 133.9, 133.8, 133.3, 129.8−126.8, 99.3,
99.2, 98.5, 97.4, 95.8, 93.7, 81.1, 80.1, 79.5, 78.8, 77.9, 75.70, 75.6, 75.2,
74.5, 74.4, 73.4, 72.9, 72.4, 71.5, 71.2, 70.0, 69.4, 69.1, 68.5, 68.4, 68.1,
67.94, 67.85, 67.3, 67.1, 60.9, 60.7, 51.5, 51.4, 50.9, 33.8, 29.0, 25.6, 24.6,
22.9, 22.4, 21.2, 20.61, 20.59, 20.5, 20.4, 18.18, 18.16, 17.9, 17.5. TOF-
+
MS-ES+ m/z calcd for [C120H138N2O38]NH4 : 2232.9. Found: 2232.9.
5-(Methoxycarbonyl)pentyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-D-
glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-
glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyra-
nosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→
2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acetamido-
4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-
4-O-benzyl-α-L-rhamnopyranoside (35). To a solution of compounds
28 (1.19 g, 0.9 mmol) and 32 (650 mg, 0.4 mmol) in CH2Cl2 (10 mL)
was added TMSOTf (2 μL) under ice-cooling. After 40 min the solution
was allowed to reach room temperature in approximately 40 min. The
solution was treated with Et3N (0.1 mL). The syrupy residue obtained
after removal of the volatiles under reduced pressure was purified by
Q
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
column chromatography using hexanes−EtOAc 2:1 → 4:3 as the eluant
afforded unreacted 32 (344 mg) followed by 35 (320 mg, 60% based on
recovered 32: 1H NMR (CDCl3, partial) δ 8.15−7.95 (m, 7H), 7.65−
7.45 (m, 10H), 7.30−6.95 (m, 43H), 5.77, 5.70 (2 d, 2 × 1H, J ∼ 9.7
Hz), 5.62 (br s, 2H), 5.42 (br m, 2H), 5.30 (dd, 1H, J = 3.6 Hz, J = 9.5
Hz), 5.24 (t, 1H, J = 9.6 Hz), 4.86 (d, 1H, J = 3.5 Hz), 3.65 (s, 3H), 2.35
(t, 2H, J = 7.5 Hz), 2.05, 1.98, 1.96, 1.93, 1.88, 1.77 (6 s, 6 × 3H); 13C
NMR (CDCl3) δ 174.0,173.9, 171.2, 171.0, 170.6, 170.1, 169.9, 169.2,
168.8, 165.6, 165.4, 165.2, 138.3, 138.2, 138.0, 137.9, 137.8, 137.6, 137.5,
137.3, 137.1, 133.9, 133.7, 133.2, 129.8−126.5, 100.0, 99.4, 98.7, 98.5,
97.6, 96.7, 94.9, 81.3, 80.1, 80.0, 79.6, 79.5, 78.8, 77.9, 75.7, 75.6, 75.2,
75.0, 74.7, 74.5, 74.4, 74.1, 73.9, 73.5, 72.8, 72.6, 72.4, 71.5, 71.2, 70.1,
69.4, 69.1, 68.5, 68.4, 68.1, 67.94, 67.85, 67.3, 67.1, 60.8, 51.6, 51.4, 50.9,
33.8, 29.0, 25.6, 24.6, 22.9, 22.4, 21.2, 20.61, 20.59, 20.5, 20.4, 18.2, 17.9,
1.57 (m, 4H), 1.42−1.34 (M, 2H), 1.39, 1.16, 1.13, 1.11 (4 d, 4 × 3H, J ∼
6.3 Hz each); 13C NMR (CDCl3) δ 173.9, 170.6, 170.5, 170.3, 168.9,
166.1, 165.6, 165.4, 165.3, 138.21, 138.20, 138.1, 138.0, 137.7,137.59,
137.57, 137.3, 133.9, 133.2, 129.77−126.70, 99.2, 99.1, 98.8, 98.4, 98.2,
97.3, 95.8, 95.5, 81.31, 81.28, 81.2, 80.0, 79.5, 78.7, 77.9, 77.8, 76.6, 75.8,
75.59, 75.56, 75.5, 75.22, 75.18, 74.6, 74.5, 74.4, 74.3, 74.1, 74.0, 73.38,
73.35, 73.2, 72.4, 71.2, 71.1, 70.4, 69.9, 69.8, 69.6, 69.4, 69.1, 68.3, 68.2,
68.0, 67.9, 67.79, 67.75, 67.7, 60.8, 60.7, 51.5, 51.4, 33.8, 28.9, 25.5, 24.5,
23.0, 22.8, 21.1, 20.8, 20.6, 20.4, 18.1, 17.9, 17.62, 17.60. TOF-MS-ES+
m/z calcd for [C165H184N2O47]Na+: 2968.2. Found: 2968.2.
5-(Methoxycarbonyl)pentyl) (2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamno-
pyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-
(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-
(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-ben-
zoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-
α-D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-
α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamno-
pyranoside (38). A stirred solution of compounds 33 (1.0 g, 1.2 mmol)
and 37 (0.66 g, 0.23 mmol) in CH2Cl2 (10 mL) was treated with
TMSOTf (5 μL) under external cooling with ice−water. After 5 min the
cooling bath was removed, and then the solution was allowed to reach
room temperature over a period of 1 h. To the solution was added Et3N
(approximately 0.1 mL) followed by concentration under reduced
pressure and column chromatography of the resulting syrup to afford 38
(0.73 g, 88%): 1H NMR (CDCl3, partial) δ 8.12−8.06 (m, 2H), 8.03−
7.94 (m, 8H), 7.67−7.32 (m, 16H), 7.31−7.12 (m, 34H), 7.17−7.00 (m,
16H), 6.98−6.92 (m, 4H), 5.74 (t, 1H, J = 9.1 Hz), 5.54 (m, 1H), 5.39
(br s, 1H), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.5 Hz), 1.98, 1.95, 1.91, 1.88,
1.85, 1.82, 1.77, 1.75, 1.67, 1.45 (10 s, 10 × 3H), 1.68−1.56 (m, 4H),
1.44−1.40 (m, 2H), 1.38, 1.22, 1.00 (3 d, 3 × 3H, J = 6.3 each), 1.25 (d,
6H, J = 6.3 Hz). 13C NMR (CDCl3) δ 174.1, 171.2, 170.7, 170.63,
170.57, 170.5, 170.4, 170.0, 169.1, 169.0, 168.9, 165.7, 165.5, 165.45,
165.41, 165.1, 138.45, 138.36, 138.24, 138.22, 137.82, 137.79, 137.74,
137.73, 137.68, 137.49, 137.45, 134.0, 133.9, 133.3, 129.9−126.8, 99.5,
99.27, 99.25, 99.0, 98.7, 98.4, 97.5, 96.2, 95.8, 93.8, 93.6, 93.2, 81.3, 81.2,
80.24, 80.20, 79.7, 79.6, 78.9, 78.0, 77.9, 75.7, 75.5, 75.3, 74.63, 74.56,
74.5, 74.2, 73.5, 73.4, 73.0, 72.7, 72.52, 72.45, 71.6, 71.3, 71.2, 70.1, 70.0,
69.7, 69.5, 69.3, 69.1, 68.9, 68.7, 68.5, 68.3, 68.1, 68.0, 67.9, 67.8, 67.7,
67.6, 67.4, 67.2, 60.9, 60.8, 60.7, 51.5, 51.4, 51.2, 51.0, 33.9, 29.0, 25.7,
24.6, 23.1, 23.0, 22.5, 22.4, 21.27, 21.26, 20.73, 20.70, 20.68, 20.59,
20.56, 20.5, 20.4, 18.3, 18.2, 18.1, 18.0, 17.7, 17.5. TOF-MS-ES+ m/z
calcd for [C199H223N3O60]Na+: 3637.2. Found: 3637.4.
5-(Methoxycarbonyl)pentyl) (2-O-acetyl-3,4,6-tri-O-benzyl-α-D-
glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-
glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyra-
nosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→
2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acetamido-
4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-
4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-
α-L-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyrano-
syl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyrano-
syl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (39).
To a stirred solution of 28 (1.00 g, 0.82 mmol) and 37 (0.66 g, 0.23
mmol) in CH2Cl2 (15 mL) was added TMSOTf (5 μL) under cooling in
ice−water. After 15 min the cooling bath was removed, and then the
solution was allowed to reach room temperature in 1 h. The solution was
treated with Et3N (∼0.1 mL) followed by removal of the volatiles.
Column chromatographic purification of the residue using hexanes−
EtOAc (3:2 → 5:4) as the eluant afforded 39 (0.95 g, 91%) as an
amorphous substance, the purity of which was about 95% (NMR): 1H
NMR (CDCl3, partial) δ 8.11−8.08 (m), 8.02−7.95 (m, 7.64−7.54 (m),
7.50−7.39 (m), 7.32−6.89 (m), 5.83 (d, 1H, J = 10 Hz), 5.73 (t, 2 × 1H,
J = 9.3 Hz), 5.55, 5.54 (2 d, 2 × 1H, J = 3.5 Hz each), 5.42−5.38 (2 m, 2
× 1H), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.5 Hz), 1.96, 1.95, 1.93, 1.88, 1.85,
1.82, 1.75, 1.71, 1.67, 1.63 (10 s, 10 × 3H), 1.38, 1.19, 0.99 (3 d, 3 × 3 H,
J = 6.3 Hz), 1.14 (d, 6H, J = 6.3 Hz); 13C NMR (CDCl3) δ 174.0, 171.1,
170.60, 170.57, 170.5, 170.3, 170.1, 169.2, 168.9, 165.7, 165.5, 165.4,
165.0, 138.4, 138.3, 138.20, 138.16, 137.8, 137.7, 137.6, 137.44, 137.40,
134.0, 133.7, 133.29, 133.26, 129.8−125.3, 99.4, 99.3, 99.20, 98.9,98.7,
98.4, 97.4, 96.20, 95.7, 95.5, 94.2, 81.3, 81.1, 80.20, 80.16, 79.9, 79.6,
+
17.5. TOF-MS-ES+ m/z calcd for [C147H166N2O43]NH4 : 2665.1.
Found: 2665.1.
5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-3-O-chlor-
oacetyl-α-L-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-gluco-
pyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-gluco-
pyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-
(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-
(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-
O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-
benzyl-α-L-rhamnopyranoside (36). TMSOTf (7.5 μL) was added to a
stirred solution of compounds 8 (2.6 g, 1.6 mmol) and 32 (1.3 g, 0.84
mmol) in CH2Cl2 (15 mL) under ice−water cooling. After 5 min the
cooling bath was removed, and the solution was allowed to reach room
temperature. After 1 h Et3N (0.2 mL) was added followed by removal of
the volatiles. The residue was purified by column chromatography
(hexanes−EtOAc 2:1 → 3:2) to give 36 (2.2 g, 88%) as an amorphous
solid: 1H NMR (CDCl3, partial) δ 8.11, 8.09, 8.05, 8.03, 8.01, 7.99, 7.97,
7.95 (8 s, 8 × 1H), 7.66−7.57 (m, 4H), 7.52−7.43 (m, 8H), 7.32−6.94
(m, 50H), 5.79, 5.75 (2 d, 2 × 1H, J = 9.9 Hz each), 5.60−5.54 (br m,
2H), 5.40 (br s, 1H), 5.37 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz), 5.3, 5.28 (1 br
s, 2 × 1H), 5.22 (t, 1H, J = 9.7 Hz), 5.20 (br s, 1H), 5.11−5.07 (m, 2H),
4.95, 4.89 (2 d, 2 × 1H, J ∼ 3 Hz), 4.92 (d, 1H, J = 11.0 Hz), 4.82 (d, 1H,
J = 11.0 Hz), 4.81 (br s, 1H), 4.17 (dd, 1H, J = 2.9 Hz, J = 9.6 Hz), 4.09
(d, 1H, J = 11.0 Hz), 3.66 (s, 3H), 2.33 (t, 2H, J = 9.5 Hz), 2.06, 2.00,
1.96, 1.84, 1.83, 1.67 (6 s, 6 × 3H), 1.66−1.56 (m, 4H), 1.42−1.34 (m,
2H), 1.39, 1.17, 1.12, 0.93 (4 d, 4 × 3H, J = 6.3 each); 13C NMR
(CDCl3) δ 173.9, 170.6, 170.5, 170.3, 169.4, 168.9, 165.9, 165.6, 165.6,
165.5, 165.4, 165.2, 138.3, 138.24, 138.15, 137.9, 137.7, 137.6, 137.38,
137.35, 133.9, 133.4, 133.3, 129.7−126.8, 100.0, 99.5, 99.2, 98.8, 98.2,
97.3, 96.7, 95.6, 81.3, 80.8, 80.0, 79.5, 79.3, 78.8, 78.4, 78.1, 77.92, 77.89,
75.7, 75.6, 75.3, 75.2, 74.9, 74.5, 74.4, 74.3, 74.1, 73.9, 73.42, 73.39, 72.5,
71.3, 71.1, 70.3, 70.1, 69.8, 69.6, 69.5, 68.4, 68.3, 68.2, 68.0, 67.9, 67.83,
67.79, 67.7, 60.7, 51.7, 51.4, 40.4, 33.8, 28.9, 25.5, 24.5, 23.1, 22.9, 21.1,
21.0, 20.7, 20.4, 18.12, 18.06, 17.6, 17.1. TOF-MS-ES+ m/z calcd for
[C167H185ClN2O48]Na+: 3044.2. Found: 3044.2.
5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-α-L-rham-
nopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→
3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→
3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-
benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-ben-
zyl-α-D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-
deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranoside (37). A mixture of compound 36 (2.14 g, 0.7
mmol), thiourea (13.2 mmol), C5H5N (1 mL), and DMF (25 mL) was
stirred at room temperature for 16 h. The solution was concentrated
under reduced pressure. To the residue CHCl3 (50 mL) was added. The
solids were removed by filtration and then were washed with CHCl3
thrice. The combined solutions were washed with brine thrice.
Concentration of the organic layer afforded a syrup that was purified
by column chromatography using hexanes−EtOAc (3:2 → 5:4) as the
eluant to afford 37 (1.6 g, 77%) as a colorless syrup: 1H NMR (CDCl3,
partial) δ 8.14−8.97 (4 m, 4 × 2H), 7.67−7.55 (m, 4H), 7.54 (m, 8H),
7.38−7.13 (m, 40H), 7.10−703 (m, 10H), 5.75 (dd, 2H, J = 9.9 Hz, J =
12.0 Hz), 5.57 (dd, 1H, J = 1.4 Hz), J = 3.5 Hz), 5.40 (m, 1H), 5.37 (dd,
1H, J = 1.5 Hz, J = 3.5 Hz), 5.32 (m, 1H), 5.29 (br s, 1H), 5.23−5.18 (m,
3H), 5.07 (dd, 1H, J = 9.1 Hz, J = 10.0 Hz), 4.95 (d, 1H, J = 3.5 Hz), 2.33
(t, 2H, J = 7.4 Hz), 1.98, 1.96, 1.90, 1.85, 1.76, 1.68 (6 s, 6 × 3H), 1.67−
R
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
79.0, 78.0, 77.9, 77.5, 76.6, 75.73, 75.70, 75.6, 75.3, 74.6, 74.5, 74.4, 74.3,
7.35, 73.4, 73.2, 73.0, 72.8, 72.5, 72.4, 71.20, 71.16, 70.5, 70.3, 70.1, 70.0,
69.5, 69.2, 68.9, 68.6, 68.4, 68.1, 68.04, 67.99, 67.9, 67.7, 60.78, 60.76,
53.4, 51.5, 51.4, 50.9, 33.9, 31.6, 29.0, 25.6, 24.6, 23.0, 22.9, 22.7, 22.6,
21.4, 21.24, 21.21, 20.8, 20.70, 20.68, 20.53, 20.51, 20.2, 18.2, 18.1, 17.9,
17.7, 17.4, 14.2, 14.1, 11.4. TOF-MS-ES+ m/z calcd for
[C226H251N3O65]Na+: 4069.5. Found: 4069.6.
Conjugation of Oligosaccharides. To 15 mg of BSA or rDT
(H21G)27 in 2.2 mL of Buffer A (PBS, 0.1% glycerol, 5 mM EDTA, pH
7.2), 4 mg of N-succinimidyl 3-(bromoacetamido) propionate in 40 μL
of DMSO was added at pH 7.2, at rt with mixing. After 1.5 h, the solution
was applied to a Sephadex G-50 column (1 × 50 cm) in PBS. The void
volume fraction containing bromoacetylated protein (Pr−Br) was
concentrated using an Amicon Ultra-15 centrifuge filter device to 2.6
mL. 13 mg of the protein was recovered, and 0.1 mL of the solution was
removed for analysis. To 12 mg of Pr−Br in 2.4 mL of Buffer A, 10 mg of
O-(3-thiopropyl)hydroxylamine were added in 300 μL of 1 M KCl,
reacted at pH 7.2, rt with mixing for 3 h. Next, the solution was passed
through Sephadex G-50 (1 × 50 cm), and the void volume fraction
containing aminooxylated protein (Pr-ONH2) was concentrated to 2.6
mL as above, and 0.2 mL removed for analysis. Pr-ONH2 (10 mg) was
treated with the oligosaccharide (10 mg) in 3 mL of Buffer A at pH 7.2 at
rt with mixing for 12 h. The solution was then passed through Sephadex
G-50 in PBS, and the void volume fraction was collected and analyzed
for sugar and protein contents and molecular mass.
Analytical Methods. Protein concentration was measured by the
BCA protein assay according to the manufacturer’s protocol. Sugar was
quantitated by the anthrone assay.43 SDS-PAGE and Western blot were
performed using standard protocols. LPS (2 μg) was loaded into 14%
Tris-Glycine gels and transferred into polyvinylidene (PDVF)
membranes according to the manufacturer’s instructions (Bio-Rad,
Hercules, CA). After the transfer, the membranes were blocked with 1%
BSA in PBS and incubated for 3 h with anti-SD or anti-E. coli O148 sera
raised by whole bacteria or by the synthetic oligosaccharide/protein
conjugates diluted 1:100 in blocking buffer. After washing 3 × 15 min
with PBS + 0.05% Tween, the membranes were incubated for 1 h with
phosphatase labeled goat antimouse IgG (KPL, Gaithersburg, MD)
diluted 1:500 in blocking buffer, washed again as above, and visualized
using BCIP/NBT phosphate substrate (KPL, Gaithersburg, MD). For
dot-blot analyses, 2 μg of conjugates were pipetted onto stripes of a
nitrocellulose membrane and developed the same way as the Western
Blot. MALDI-TOF mass spectra of the derivatized proteins and of the
conjugates were obtained with a MALDI-TOF instrument operated in
the linear mode. Samples for analysis were desalted, and a 1 μL aliquot
was mixed with 20 μL of a saturated sinapinic acid matrix solution made
in 30% aq CH3CN containing 0.1% trifluoroacetic acid. One microliter
of the solution so obtained was applied to and dried on the sample stage.
Immunization. All animal experiments were approved by the
National Institute of Child Health and Human Development Animal
Care and Use Committee. Five to six-week-old female NIH general
purpose mice were immunized subcutaneously 3 times at 2 week
intervals, with 2.5 μg of oligosaccharide as a conjugate in 0.1 mL of
phosphate buffered saline (PBS). Groups of 10 mice were exsanguinated
7 days after the third injection.44 Controls received PBS.
5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-3-O-chlor-
oacetyl-α-L-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-gluco-
pyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-gluco-
pyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-
(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl)-(1→2)-
(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-
O-acetyl-2-deoxy-α-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-
benzyl-α-L-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-
rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-D-glucopyranosyl)-
(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-D-glucopyranosyl)-
(1→3)-(2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranoside (40). To a
stirred solution of imidate 8 (1.6 g, 0.98 mmol) and octasaccharide 37
(1.5 g, 0.52 mmol) in CH2Cl2 (15 mL) was added TMSOTf (7.5 μL)
under cooling in ice−water. The solution was allowed to reach room
temperature. After 1 h the solution was treated with Et3N (0.1 mL)
followed by concentration. Repeated column chromatography of the
residue using hexanes−EtOAc (3:2 → 6:5) as the eluant afforded 40
(1.45 g, 64%) as an amorphous material: 1H NMR (CDCl3, partial) δ
8.15−7.97 (m, 12H), 7.7−7.57 (m, 7H), 7.55−7.43 (m, 14H), 7.35−
7.21 (m, 46H), 7.21−6.96 (m, 26H), 3.69 (s, 3H), 5.83−5.74 (m, 3H),
5.63−5.55 (m, 3H), 5.43 (br s, 1H), 5.39 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz),
5.34 (br s, 1H), 5.29 (br s, 3H), 5.25−5.19 (m, 3H), 5.16−5.09 (m, 3H),
4.97 (d, 1H, J = 3.2 Hz), 2.36 (t, 4H, J = 7.5 Hz), 2.09, 2.01, 1.98, 1.90,
1.88, 1.86, 1.82, 1.77, 1.70 (9 s, 9 × 3H), 1.70−1.60 (m, 4H), 1.43−1.37
(m, 2H), 1.41, 1.19, 1.18, 1.12, 1.09, 0.95 (6 d, 6 × 3H, J = 6.3 each); 13C
NMR (CDCl3) δ 174.0, 170.64, 170.62, 170.58, 170.5, 170.4, 170.3,
169.4, 168.9, 166.0, 165.6, 165.52, 165.46, 165.44, 165.42, 165.3, 165.2,
138.36, 138.35, 138.3, 138.2, 138.23, 138.16, 138.0, 137.80, 137.75,
137.70, 137.69, 137.67, 137.5, 137.43, 137.41, 137.39, 137.38, 133.93,
133.87, 133.4, 133.3, 133.2, 129.8 − 126.8, 100.1, 99.6, 99.2, 98.9, 98.3,
97.4, 96.8, 95.9, 95.7, 81.28, 81.25, 80.8, 80.2, 80.1, 79.6, 79.40, 78.9,
78.8, 78.4, 78.1, 77.9, 75.72,75.67, 75.65, 75.4, 75.2, 74.9, 74.6, 74.5, 74.4,
74.2, 74.1, 73.9, 73.45, 73.43, 73.38, 72.5, 72.4, 71.3, 71.2, 71.1, 70.4,
70.2, 69.9, 69.6, 69.5, 69.4, 68.44, 68.39, 68.2, 68.1, 68.0, 67.9, 67.8,
67.72, 67.68, 60.8, 60.65, 51.7, 51.5, 51.3, 25.6, 24.6, 23.2, 23.0, 22.9,
21.2, 21.1, 21.0, 20.7, 20.5, 18.2, 18.1, 18.0, 17.6, 17.5, 17.1. TOF-MS-
ES+ m/z calcd for [C246H270ClN3O70]Na+: 4443.7. Found: 4444.0.
5-Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-glu-
copyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→
3)-α-L-rhamnopyranoside (41). For 1H and 13C NMR data, see Tables
1, 3, 5, and 7.
5-Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-glu-
copyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→
3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-
glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-
1
(1→3)-α-L-rhamnopyranoside (42). For H and 13C NMR data, see
Serology. IgG antibodies were measured by ELISA using BSA or
human serum albumin as appropriate for blocking. Antibody levels were
calculated relative to a pool of highest antibody level sera obtained from
mice immunized 3 times with S. dysenteriae type 1 conjugates14 and
assigned a value of 100 ELISA units (EU). Results were computed with
an ELISA immunoassay data processing program provided by the
Biostatistics and Information Management Branch, CDC.45 Compet-
itive inhibition ELISA was done by incubating sera from mice injected
with either S. dysenteriae type 1 or E. coli O148 synthetic 12-mers
conjugated to rDT, diluted in PBS to give an A405 of 1.0, with 0.04, 0.2, 5,
or 80 μg/well of either O-SP, incubated for 1 h at 37 °C, followed by
incubation at 4 °C for 12 h. The assay was then continued as described
above. Sera with inhibitor were compared to the same serum dilution
without an inhibitor. Percent inhibition was defined as (1-A405 adsorbed
serum/A405 nonadsorbed serum) × 100%.
Tables 1, 3, 5, and 7.
Growth of Bacteria, Isolation of LPS and O-SP. E. coli O148
strain 201 and rabbit typing antiserum were obtained from the Centers
for Disease Control and Prevention (Atlanta, GA) and cultured in
tryptic soy broth for 20 h at 37 °C with stirring and aeration. The pH was
maintained at ∼7.5 by the addition of ammonium hydroxide. The
identity of the bacteria was confirmed by the Clinical Microbiology
Laboratory at the NIH, Bethesda, MD. LPS was extracted by the hot
phenol method.42 LPS was purified by ultracentrifugation in a Sorvall
Discovery SE centrifuge twice at 35 000 rpm for 5 h, at 4 °C. The
contents of proteins and nucleic acids in the final LPS preparation were
less than 1.5%. To isolate O-SP, LPS (100 mg) was treated with 10 mL
of 1% acetic acid at 100 °C for 1.5 h. Lipid A was removed by
ultracentrifugation as above, and the soluble products were separated by
gel chromatography on a BioGel P-10 (1 × 100 cm) column equilibrated
with 0.05 M pyridinium acetate buffer at pH 5.5. The chromatography
was monitored with a differential refractometer. O-SP was eluted in the
void volume fraction with a yield of 28%.
Statistics. ELISA values are expressed as the geometric mean (GM).
Unpaired t-tests were used to compare GMs of different groups.
S
dx.doi.org/10.1021/jo300299p | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(23) Roy, R.; Shiao, T. C. Chimia 2011, 65, 24−29.
ASSOCIATED CONTENT
■
(24) Huang, Y. L.; Wun, C. Y. Expert Rev. Vaccines 2010, 9, 1257−
S
* Supporting Information
1274.
1H and 13C NMR spectra for compounds 1−8, 10, 13, 14, 18−
30, 32, and 34−42. This material is available free of charge via the
Internet at http://pubs.acs.org/.
(25) Pozsgay, V.; Kubler-Kielb, J. Synthesis of carbohydrate antigens
related to Shigella dysenteriae type 1 and of their protein conjugates. In
Frontiers in Carbohydrate Chemistry; Demchenko, A. V., Ed.; American
Chemical Society: Washington, DC, 2006; pp 238−252.
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Pozsgay, V.; Shiloach, J.; Schneerson, R. Proc. Natl. Acad. Sci. U. S. A.
2009, 106, 7974−7978.
(29) Pozsgay, V.; Ekborg, G.; Sampathkumar, S.-G. Carbohydr. Res.
2006, 341, 1408−1427.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +1 301 295 2266. Fax: +1 301 295 1435. E-mail:
pozsgayv@mail.nih.gov.
■
Notes
The authors declare no competing financial interest.
(30) Kubler-Kielb, J.; Pozsgay, V. J. Org. Chem. 2005, 70, 6987−6990.
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1989, 191, 29−52.
ACKNOWLEDGMENTS
■
This work was supported by the intramural programs of the
Eunice Kennedy Shriver National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda,
MD. The authors thank Noel Whittaker for the mass spectra.
(33) Pozsgay, V.; Jennings, H. J. Synthesis 1990, 724−726.
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