Chemical synthesis and immunological evaluation of the inner core oligosaccharide of francisella tularensis

Article abstract of DOI:10.1021/ja306274v

Francisella tularensis, which is a Gram negative bacterium that causes tularemia, has been classified by the Center for Disease Control and Prevention (CDC) as a category A bioweapon. The development of vaccines, immunotherapeutics, and diagnostics for F. tularensis requires a detailed knowledge of the saccharide structures that can be recognized by protective antibodies. We have synthesized the inner core region of the lipopolysaccharide (LPS) of F. tularensis to probe antigenic responses elicited by a live and subunit vaccine. The successful preparation of the target compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A comparison of the ¹H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment of the inner core oligosaccharide of F. tularensis. The observation that immunizations with LPS lead to antibody responses to the inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.

Full text of DOI:10.1021/ja306274v

Article
pubs.acs.org/JACS
Chemical Synthesis and Immunological Evaluation of the Inner Core
Oligosaccharide of Francisella tularensis
Thomas J. Boltje,† Wei Zhong,† Jin Park,† Margreet A. Wolfert,† Wangxue Chen,‡ and Geert-Jan Boons*,†
†Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States
‡Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada
S
* Supporting Information
ABSTRACT: Francisella tularensis, which is a Gram negative
bacterium that causes tularemia, has been classified by the
Center for Disease Control and Prevention (CDC) as a
category A bioweapon. The development of vaccines,
immunotherapeutics, and diagnostics for F. tularensis requires
a detailed knowledge of the saccharide structures that can be
recognized by protective antibodies. We have synthesized the
inner core region of the lipopolysaccharide (LPS) of F.
tularensis to probe antigenic responses elicited by a live and
subunit vaccine. The successful preparation of the target
compound relied on the use of a disaccharide which was modified by the orthogonal protecting groups diethylisopropylsilyl
(DEIPS), 2-naphthylmethyl (Nap), allyl ether (All), and levulinoyl (Lev) ester. The ability to remove the protecting groups in
different orders made it possible to establish the optimal glycosylations sequence to prepare a highly crowded 1,2,3-cis configured
branching point. A variety of different methods were exploited to control anomeric selectivities of the glycosylations. A
comparison of the 1H NMR spectra of isolated material and the synthetic derivative confirmed the reported structural assignment
of the inner core oligosaccharide of F. tularensis. The observation that immunizations with LPS lead to antibody responses to the
inner core saccharides provides an impetus to further explore this compound as a vaccine candidate.
component such as a protein antigen or capsular and
lipopolysaccharides (LPS).5 In particular, LPS-based vaccines
are attractive, and for example, it has been shown that mice
vaccinated with the O-antigen released by mild acid hydrolysis
of LPS and conjugated to BSA can protect against an
intradermal challenge with a highly virulent type B strain of
F. tularensis, and partially protect against an aerosol challenge
with the same strain.6 It has also been shown that mice
intradermally inoculated with intact LPS from F. tularensis
acquire varying degrees of resistance against systematic or
aerogenic challenge with virulent strains of the pathogen.7
More recently, it was found that a detoxified LPS complex with
an outer membrane protein of N. meningitidis group B can
protect mice against a lethal respiratory challenge with the
highly virulent F. tularensis SchuS4.8
The structure of LPS of F. tularensis has been determined,
and it contains a lipid A moiety, a core oligosaccharide, and an
O-chain polysaccharide. The O-antigen is composed of
tetrasaccharide repeating units, which consist of two N-acetyl
galactosamine uronamides and an N-acetyl quinovosamine and
N-formyl-4-amino-quinovose moiety.9 Furthermore, structural
studies have shown that the core region has a highly unusual
composition.10 It is linked to the lipid A region by only one 3-
deoxy-D-manno-2-octulosonic acid (KDO) moiety (A) instead
INTRODUCTION
■
Francisella tularensis is the etiologic agent of tularemia (rabbit
fever) in humans and animals.1 It is a Gram-negative,
facultative, intracellular pathogen that can survive and
propagate within phagocytic cells. In nature, a disease cycle is
maintained between wild animals such as rabbits, beavers,
squirrels, and water rats and biting vectors such as flies, ticks,
mosquitoes, and mites and the contaminated environment.2 F.
tularensis is highly virulent, requiring as few as 10−50 cells to
cause human infection.3 It can survive for long periods of time
under harsh environmental conditions. Tularemia may occur in
different forms but the pneumonic form is associated with the
highest mortality (30% without antibiotic treatment). F.
tularensis has been classified by the Center for Disease Control
and Prevention (CDC) as a top-priority (Category A)
bioterrorism agent. Common to all Category-A select agents,
F. tularensis transmits easily, has the capacity to inflict
substantial morbidity and mortality on a large number of
people and can induce widespread panic.4 Aerosol dispersal is
considered the most hazardous mode of transmission, as it
would affect the most people.
To prevent infections by F. tularensis, an attenuated live
vaccine strain (LVS) was developed in the 1950s, but was not
licensed for use as a human vaccine in the United States
because the nature of its attenuation was not known and may
not be stable. Considerable efforts are being expended to
develop a subunit vaccine composed of a cell surface
Received: July 3, 2012
Published: August 6, 2012
© 2012 American Chemical Society
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is undesirable. It is also difficult to conjugate short
oligosaccharides to carrier proteins without destroying vital
immunological domains. Synthetic chemistry can address these
issues since it makes it possible to incorporate an artificial linker
for controlled conjugation to proteins.12 Furthermore, it can
provide substructures for establishing structure−activity
relationships or can be used to determine minimal epitope
requirements to elicit protective immune responses.
Herein, we report the synthesis of the complete hexasac-
charide inner core domain of F. tularensis LPS and the
preparation of biotin and protein conjugates thereof. Immune
recognition of the hexasaccharide by antisera of mice
immunized with a live-attenuated vaccine or LPS has been
determined.
RESULTS AND DISCUSSION
■
The chemical synthesis of hexasaccharide 1 is challenging due
to its highly branched nature, which complicates the installation
of the various glycosidic linkages. Furthermore, the target
compound contains a number of glycosides that are difficult to
install in a stereoselective fashion and in particular the
introduction of β-mannosides, α-glucosides, and α-linked
galactosamines often leads to the formation of a mixture of
anomers, which may be difficult to separate and lower the yield
of required products.13 Furthermore, hexasaccharide 1 has a
free amine and carboxylic acid, which makes conjugation to
protein carriers or biotin challenging (compounds 2 and 3).
The latter type of conjugation is, however, required for
immunological evaluations.
It was envisaged that disaccharide 4 which at C-1, C-2, C-2′,
and C-3′ is modified by the orthogonal protecting groups allyl
ether (All), levulinoyl (Lev) ester, diethylisopropylsilyl
(DEIPS) and 2-methylnaphthyl (Nap), respectively, would
provide a flexible intermediate to prepare the target
compound.14 The orthogonal protecting groups made it
possible to establish the optimal sequence of glycosylation to
install the highly crowded branching points. It also minimized
protecting group manipulations during oligosaccharide assem-
bly and offers future opportunities to synthesize a library of
structurally related oligosaccharides for immunological studies.
The α-linked 2-amino-2-deoxy-galactoside of 1 could be
installed by using glycosyl donor 5 or 6 which are modified by a
4,6-O-di-tert-butylsilyl acetal, which sterically blocks the β-face
thereby providing only an α-linked galactoside even in the
presence of a C-2 participating group.15 Glycosyl donors 7 and
8 were prepared to explore the stereoselective introduction of
the α-glucoside moiety of 1. In particular, compound 7 was
deemed attractive because the C-2 (S)-(phenylthiomethyl)-
benzyl ether can perform neighboring group participation
Figure 1. Target hexasaccharide 1 and the monosaccharide building
blocks required for its assembly.
of the usual two KDO residues (Figure 1). It does not contain
heptosyl residues but contains two mannosyl moieties. One of
the mannosides (C) is β-linked to another mannoside (B), and
this disaccharide fragment is further substituted at C-2, C-2′,
and C-3′ by a β-glucoside (E), an α-galactosamine (D), and an
α-glucoside (F), respectively.
The development of vaccines, immunotherapeutics, and
diagnostics for F. tularensis requires a detailed knowledge of the
saccharide structures that can be recognized by protective
antibodies. It also needs well-defined oligosaccharides con-
jugated to carrier proteins for immunizations to establish
structural motifs that can provide protection. Although
oligosaccharide fragments can be obtained by controlled
hydrolysis of LPS,11 chemical synthesis offers a much more
attractive approach to obtain such compounds.12 Obviously,
isolation of oligosaccharides from a Class A bioterrorism agent
a
Scheme 1. Preparation of KDO Building Block 11
a
Reagents and conditions: a) DAST, CH2Cl2, −50 °C, 15 min, 50% b) N-benzyloxycarbonyl-3-amino-propanol, BF3·Et2O, CH2Cl2, 0 °C, 1 h, 55%
c) i. AcOH, H2O, reflux, 1 h, 96% ii. 2-methoxy propene, p-TsOH, 1,4-dioxane, DMF, rt, 16 h, 85% d) Bu2SnO, MeOH, reflux, 3 h, then BnBr, CsF,
DMF, rt, 16 h, 84%.
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during glycosylation to give an intermediate anomeric β-
sulfonium ion, which upon displacement by a sugar alcohol will
selectively provide an α-glucoside.16 In the case of compound
8,17 which carries a nonparticipating benzyl ether at C-2,
solvent effects will need to be exploited to control α-anomeric
selectivity.18 Installation of the β-glucoside of 1 should be
straightforward by employing trifluoro-N-phenylacetimidates
919 or 10,20 which have an acetyl ester at C-2 that participates
during the glycosylation to give selectively the required β-
anomer. Finally, KDO building block 11 carries an aminopropyl
linker at its anomeric center, which was expected to allow
conjugation to a carrier protein or biotin moiety.
Scheme 2. Assembly of the Trisubstituted β-Mannoside
Core
a
The preparation of KDO building block 11 commenced with
known derivative 12,21 which was treated with diethylamino-
sulfur trifluoride (DAST) to afford glycosyl fluoride 13 in a
moderate yield due to the formation of a 2,3-unsaturated
byproduct (Scheme 1). Glycosylation of 13 with benzylox-
ycarbonyl protected aminopropanol in the presence of
BF3·Et2O as the promoter led to the formation of 14 as a
separable mixture of α/β anomers (α/β = 3/1). The
isopropylidene acetals of 14 were hydrolyzed using a mixture
of acetic acid and water and the exocyclic diol of the resulting
compound was selectively reprotected as an isopropylidene
acetal22 using 2-methoxy-propene and a catalytic amount of p-
toluenesulfonic acid (p-TsOH) in DMF to give diol 15. The
equatorial alcohol of the latter derivative was selectively
benzylated by first forming an intermediate stannyl acetal
which was treated with benzyl bromide in the presence of CsF
to give the required acceptor 11.22
Next, attention was focused on the preparation of the
protected β-D-Man-(1→4)-D-Man disaccharide 4. β-Manno-
sides, which are an important class of 1,2-cis glycosides, are
difficult to introduce due to the axial C-2 substituent, which
sterically blocks incoming nucleophiles from the β-face and the
Δ-anomeric effect, which provides additional stabilization of the
α-anomer.23 Crich and co-workers have pioneered an attractive
approach for the introduction of β-mannosides by in situ
formation of an intermediate α-anomeric triflate because of a
strong endoanomeric effect.24 An SN2 like-displacement of the
α-triflate by a sugar hydroxyl will then result in the formation of
a β-mannoside. A prerequisite of β-mannoside formation is that
the donor is protected by a 4,6-O-benzylidene acetal. It has
been proposed that this protecting group opposes oxacarbe-
nium formation (SN1 glycosylation) due to the torsional strain
engendered by the half chair or boat conformation of this
intermediate and a destabilizing electronic effect caused by
placing the O-6 dipole antiparallel to the oxacarbenium ion.25
Thus, low-temperature activation of 16 with p-nitrobenzene-
sulfenyl chloride26 and silver trifluoromethanesulfonate
(AgOTf) was complete within minutes and subsequent
addition of glycosyl acceptor 17 led to the clean formation of
β-mannoside 4 as mainly the β-anomer (β/α = >20/1). The
use of trifluoromethanesulfonic anhydride (Tf2O) and 1-
benzenesulfinylpiperidine27 (BSP) as the promoter system led
to significant lower yields of product.
a
Reagents and conditions: a) 16, p-NO2C6H4SCl, AgOTf, DTBMP, 5
min, −78 °C then 17, 3 h, −78 °C→ −35 °C, 73%. b) DDQ, CH2Cl2,
H2O, 3 h, rt, (93%, 18), (72%, 23). c) TBAF, AcOH, THF, 16 h, rt,
(98%, 19), (81%, 21) d) 7, TfOH, CH2Cl2, 30 min, −35 °C→0 °C
then 18, DTBMP, 16 h, −35 °C→rt, 73%. e) NIS, TfOH, CH2Cl2, 10
min, 0 °C, 77%. f) 5 and 21, NIS, TfOH, CH2Cl2, 10 min, 0 °C,
∼10%. 6 and 21, AgOTf, DTBMP, CH2Cl2, 30 min, 0 °C, ∼10%. 7,
TfOH, CH2Cl2, 30 min, −35 °C→0 °C then 23, DTBMP, 16 h, −35
°C→rt, ∼10%. g) 8, TfOH, Et2O, 10 min, −35 °C, 72%.
the use of TBAF buffered with acetic acid led to clean
formation of alcohol 19 in 98% yield.14
Having glycosyl acceptors 18 and 19 at hand, attention was
focused on the installation of the α-glucoside and α-
galactosamine moieties. Preactivated glycosyl donor 7 with
TfOH to form an intermediate sulfonium ion followed by the
addition of glycosyl acceptor 18 gave trisaccharide 20 in a good
yield of 73% as only the α-anomer. Alternatively, a
glycosylation of 19 with 5 in the presence of NIS30 and triflic
acid (TfOH) afforded trisaccharide 22 in a yield of 77% as only
the α-anomer. The trisaccharides 20 and 22 were converted
into glycosyl acceptors by removal of the DEIPS and Nap ether
using the aforementioned conditions to give glycosyl acceptors
21 and 23, respectively.
To explore the installation of the α-glucoside and α-
galactosamine moieties, optimal conditions for the removal of
the 2-naphthylmethyl and diethylisopropylsilyl ether needed to
be established. The Nap ether of 4 could be readily removed by
oxidation with DDQ in wet DCM to give compound 18 in a
yield of 93%.14,28 Treatment of 4 with TBAF to cleave the
DEIPS ether29 led to partial removal of the Lev ester; however,
Extension of trisaccharide acceptors 21 and 23 to give
tetrasaccharide 24 proved to be challenging. Thus, a
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a
Scheme 3. Synthesis of the Inner Core of F. tularensis (Compounds 1−3)
a
Reagents and conditions: a) i. Pd(PPh3)3, AcOH, CH2Cl2, 3 h, rt then ii) TCA, DBU, CH2Cl2, 1 h, rt, 75%. b) 11, TfOH, CH2Cl2, 10 min, 0 °C,
61%. c) N2H4·AcOH, EtOH, toluene, 30 min, rt, 78%. d) 9, TfOH, CH2Cl2, 10 min, 0 °C, 82%. e) DDQ, CH2Cl2, H2O, 3 h, rt, 76%. f) 8, TfOH,
Et2O, 10 min, −35 °C, 73%. g) i). TFA, CH2Cl2, H2O, 1 h, rt, then ii) HF·pyridine, THF, 30 min, rt, then iii) Zn, AcOH, CH2Cl2, 3 h, rt, 89%, then
NaOMe, MeOH, H2O, THF, 1 h, rt, 55%, then v) Pd(OH)2, H2, t-BuOH, H2O, 16 h, rt, 85%. h) PBS pH 7.4, Biotin-OSu, 16 h, rt, 62%. i) SAMA-
Opfp, DIPEA, DMF, 3 h, rt, 53%.
glycosylation of 21 with glycosyl donor 5 using the
aforementioned conditions led to a low yield of tetrasaccharide
24 and extensive decomposition of the glycosyl donor and
acceptor was observed (Scheme 2). Mass spectrometric analysis
of the crude product showed cleavage of the (S)-
(phenylthiomethyl)benzyl ether of 21 which probably arose
from reaction with the thiophilic iodonium ion promoter.
Therefore, the glycosylation was repeated using glycosyl
bromide 6 which can be activated under mild conditions that
were expected to be compatible with the (S)-
(phenylthiomethyl)benzyl ether. Coupling of 6 with 21 in
the presence of AgOTf and DTBMP gave fewer byproducts;
however, tetrasaccharide 24 was still isolated in a low yield
(Scheme 2).
The distal mannoside of 24 is glycosylated at C-1, C-2, and
C-3, which are oriented in a 1,2,3-cis configuration rendering
the bisecting C-2 alcohol inaccessible when C-1 and C-3 are
glycosylated. Therefore, the preparation of tetrasaccharide 24
was examined by glucosylation of the C-3 hydroxyl of 23. Thus,
low temperature activation of glycosyl donor 7 with TfOH to
form an intermediate sulfonium ion was complete within
minutes; however, addition of trisaccharide acceptor 23 led
only to the formation of a small amount of tetrasaccharide 24
(∼10%), and mainly glycosyl acceptor 23 was recovered. The
failure of the glycosylation may be due to the bulky nature of
the intermediate sulfonium ion, which may not be able to react
with a sterically hindered alcohol. To test this hypothesis,
trisaccharide 23 was coupled with glycosyl donor 8 using a
catalytic amount of TfOH in diethyl ether, and fortunately
these reaction conditions afforded tetrasaccharide 25 in yield of
72% as mainly the α-anomer (α/β = 20/1). Probably, glycosyl
donor 8 reacts through a solvent-stabilized oxa-carbenium ion,
which is more reactive and less sterically demanding than the
corresponding β-sulfonium ion of 7.
Having established the proper order for the introduction of
the α-glucoside and α-galactosamine moieites, attention was
focused on the further addition of glycosyl residues to 22
(Scheme 3). Due to the flexibility of our approach, this
elaboration can take place in a number of different ways, but
since the Lev ester is needed for the stereoselective
introduction of the α-(1→5)-mannosyl linkage to KDO, this
glycosylation was undertaken first. Thus, the anomeric allyl
moiety of trisaccharide 22 was removed using Pd(PPh3)4 in a
mixture of CH2Cl2 and AcOH, and the resulting lactol was
converted into the corresponding trichloroacetimidate 26 by
treatment with trichloroacetonitrile and 1,8-diazazdicycloun-
dec-7-ene (DBU) in DCM. A TfOH-mediated glycosylation
glycosyl donor 26 with KDO acceptor 11 in CH2Cl2 affords
tetrasaccharide 27 in a yield of 61%, and due to neighboring
group participation of the Lev ester only the β-anomer was
formed. The Lev ester of 27 could be selectively removed using
hydrazinium acetate in a mixture of toluene and ethanol
without affecting the other base sensitive functionalities to give
acceptor 28 in a yield of 78%.31 A glucosylation of
tetrasaccharide 28, with 919 having a C-2 acetyl ester to
control β-anomeric selectivity, afforded the corresponding
pentasaccharide 29 in good yield. Surprisingly, the use of
similar donor 1020 having benzyl ethers at C4 and C-6 instead
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10
1
1
Figure 2. Confirmation of structural integrity by NMR. (A) Reported H NMR spectrum of the isolated hexasaccharide fragment. (B) H NMR
1
spectrum of the synthetic hexasaccharide fragment 1. (C) H NMR spectrum of the synthetic biotin conjugate 2. (D) HMBC spectrum of 2. The
correlation between the amide carbonyl at 176.6 ppm and the CH2 signals of the linker (3.21−3.35 ppm) and biotin (2.25 ppm and 1.62 ppm)
moiety confirms that biotinylation occurred at the desired site.
of a benzylidene acetal provided a pentasaccharide in low yield.
Oxidative cleavage of the Nap ether of 29 by DDQ in a mixture
of CH2Cl2 and water afforded glycosyl acceptor 30, which was
coupled with glucosyl donor 8 under aforementioned
conditions to give the fully assembled hexasaccharide 31 (α/
β ≥ 20/1).
hydroxysuccinimido biotin (1.0 equiv) in PBS buffer (pH 7.4)
afforded a monobiotinylated product as the major reaction
product as judged by MALDI-TOF mass spectrometry and
TLC analysis. The compound was purified by reverse phase C-
18 chromatography and separated from starting material 1 and
1
a dibiotinylated derivative. H NMR analysis of 2 revealed that
The deprotection of 31 started with the removal of the
isopropylidene acetals using TFA in a mixture of CH2Cl2 and
water. It was expected that the 4,6-O-di-tert-butylsilyl acetal
would also be cleaved under these conditions; however, this
functionality proved to be remarkably stable, and therefore the
resulting diol was treated with HF·pyridine to remove the silyl
acetal, which was complete in 30 min. The resulting derivative
was treated with Zn powder in a mixture of CH2Cl2 and AcOH
to remove the Troc carbamate to afford a partially deprotected
derivative in 89% yield over three steps after purification by
LH-20 size exclusion chromatography. Next, the acetyl and
benzoyl esters were removed using NaOMe in a mixture of
MeOH/THF/H2O, and finally, hydrogenation using H2 and
Pd(OH)2 in mixture of t-BuOH and water afforded
hexasaccharide 1.
the signals adjacent to the amine had moved downfield
compared to those of 1, consistent with amide formation at this
site (Figure 2C). Furthermore, the heteronuclear multiple bond
coherence (HMBC) spectrum (Figure 2D) showed a coupling
between the amide carbonyl and the CH2 protons of the linker,
confirming the site of reaction. In addition to the biotin
derivative 2, a keyhole limpet hemocyanin (KLH) conjugate
was prepared for future immunizations. The conjugation of 1 to
KLH entailed a two-step procedure. First, hexasaccharide 1 was
reacted with perfluorophenyl 2-(acetylthio)acetate and DIPEA
in DMF to afford 3 (see Scheme 3). The regioselectivity was
again confirmed by the heteronuclear multiple bond coherence
coupling between the amide carbonyl and the CH2 protons on
the linker (see Supporting Information). Next, the thioacetyl
was cleaved using ammonia in DMF under an inert atmosphere
to prevent disulfide formation. The resulting thiol was reacted
with maleimide-activated KLH to afford the corresponding
KLH conjugate. Analysis of the KLH conjugate using high-pH
anion-exchange chromatography (HPAEC) showed that 339
glycans were present per protein molecule.
Antigenic responses against the inner core region of F.
tularensis LPS elicited by a live vaccine32 and a LPS
preparation6 were investigated. Thus, streptavidin-coated
microtiter plates were treated with biotin-modified compound
2, and serial dilutions of sera were added. Detection was
accomplished with antimouse IgG antibodies labeled with
alkaline phosphatase. No appreciable levels of IgG antibodies
were observed in the serum samples of mice immunized with
live vaccine strain. However, antibodies were detected in mice
1
The H NMR spectrum of compound 1 is in excellent
agreement with the reported 1H NMR spectrum of the isolated
LPS fragment (see A and B of Figure 2).10 In addition, the
anomeric signals of 1 displayed the appropriate chemical shifts
and homonuclear as well heteronuclear coupling constants
consistent with the desired product (see Supporting
Information). Together these findings unequivocally confirm
the reported structural assignment of the isolated LPS
fragment.
To perform immunological experiments, it was imperative to
selectively derivatize the aminopropanol linker of 1 with a
biotin moiety. It was anticipated that the amine of the artificial
spacer would be more reactive than the amine of the 2-amino-
2-deoxy galactosyl moiety of 1. Indeed, reaction of 1 with N-
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in Hertz (Hz), integration. All NMR signals were assigned on the basis
of 1H NMR, 13C NMR, COSY, and HSQC experiments. Mass spectra
were recorded on an MALDI-TOF mass spectrometer. The matrix
used was 2,5-dihydroxybenzoic acid (DHB) and Ultamark 1621 as the
internal standard. Column chromatography was performed on silica
gel G60 (Silicycle, 60−200 μm, 60 Å). TLC-analysis was conducted on
Silicagel 60 F254 (EMD Chemicals inc.) with detection by UV-
absorption (254 nm) and by spraying with 20% sulfuric acid in ethanol
followed by charring at ∼150 °C or by spraying with a solution of
(NH4)6Mo7O24·H2O (25 g/L) in 10% sulfuric acid in ethanol followed
by charring at ∼150 °C. CH2Cl2 was freshly distilled from calcium
hydride under nitrogen prior to use. Molecular sieves (4 Å) were flame
activated under vacuum prior to use. All reactions were carried out
under an argon atmosphere unless it is stated otherwise. KLH was
purchased from Thermo Fisher Scientific inc.
Figure 3. Immunoreactivity of inner core oligosaccharide (2) to
antisera elicited by a live vaccine strain (LVS) and LPS of F. tularensis.
Microtiter plates were coated with compound 2 and serial dilutions of
mouse antisera and control serum (starting dilution 1:128) were
applied to the coated microtiter plates. The optical density values are
reported as the means of triplicate measurements (see the Supporting
Information for the SD of the measurements).
2-Deoxy-2-amino-α-D-galactosamine-(1→2)-3-O-[α-D-gluco-
pyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-O-[β-D-gluco-
pyranosyl-(1→2)]-α-D-mannopyranosyl-(1→5)-3-amino-prop-
yl-3-deoxy-α-D-manno-octulopyranosidonate (1). Compound
31 (70 mg, 26 μmol) was dissolved in a mixture of CH2Cl2 (4 mL),
H2O (0.2 mL), and TFA (0.4 mL), and the resulting mixture was
stirred for 2 h at rt. Toluene (4 mL) was added, and the mixture was
concentrated in vacuo. The residue was dissolved in THF (4 mL), and
HF·Pyridine (0.7 mL) was added; the resulting mixture was stirred for
2 h at rt. EtOAc (10 mL) and sat. aq NaHCO3 (4 mL) were added
dropwise, and the organic layer was separated, dried (MgSO4), filtered,
and concentrated in vacuo. The residue was purified by LH-20 size
exclusion column chromatography (CH2Cl2/MeOH, 1/1), and the
appropriate fractions were concentrated in vacuo. The residue was
dissolved in a mixture of AcOH (2 mL) and CH2Cl2 (1 mL), and Zn
powder (30 mg) was added. The resulting suspension was stirred for 2
h at rt after which the mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by LH-20 size
exclusion column chromatography (CH2Cl2/MeOH, 1/1), and the
appropriate fractions were concentrated in vacuo. The residue (50 mg,
89% for three steps) was dissolved in mixture of MeOH (1 mL), THF
(1 mL), and water (0.3 mL), and 30% NaOMe in MeOH (0.05 mL)
was added. The resulting mixture was stirred for 2 h at rt, and AcOH
(0.1 mL) was added. The mixure was concentrated in vacuo, and the
residue was purified by reverse-phase C-18 column chromatography (0
− 90% MeOH/H2O). The appropriate fractions were collected and
concentrated in vacuo. The residue (25 mg, 55%) was dissolved in a
mixture of water (1 mL) and t-BuOH (1 mL), and Pd(OH)2 (20 mg)
was added. A hydrogen atmosphere was created, and the mixture was
stirred for 36 h at rt. The mixture was filtered, and the filtrate was
concentrated in vacuo to afford 1 (12 mg, 85%); 1H NMR (600 MHz,
D2O): δ 5.61 (d, 1H, J = 3.0 Hz, H-1-D), 5.34 (d, 1H, J = 3.0 Hz, H-1-
F), 5.14 (s, 1H, H-1-B), 4.85 (d, 1H, J = 7.8 Hz, H-1-C), 4.51 (d, 1H, J
= 7.8 Hz, H-1-E), 4.37−4,35 (m, 1H, H-2-C), 4.29−4.26 (m, 1H, H-2-
B), 4.23 (t, 1H, J = 9.0 Hz, H-5-D), 4.17−4.13 (m, 1H, H-4-A), 4.10−
3.61 (m, 29H, H-6a,b-B,C,D,E,F, H-3D, H-4-D, H-2-F, H-3-F, H-4-F,
H-5-F, H-3-B, H-4-B, H-3-C, H-4-C, H-5-C, H-3-E, H-4-E, H-5-E, H-
5-A, H-6-A, H-7-A, H-8-A), 3.52−3.33 (m, 6H, CH2 Linker, H-2-E, H-
2-D, H-3-E, H-5-B), 3.20−3.19 (m, 1H, CHH Linker), 3.08−3.04 (m,
1H, CHH Linker), 2.01 (dd, 1H, J = 4.2 Hz, J = 12.6 Hz, H-3a-A),
1.94−1.91 (m, 2H, CH2 Linker), 1.75 (t, 1H, J = 12.0 Hz, H-3b-A);
13C NMR (125 MHz, D2O) δ 175.4, 101.6, 100.2, 99.4, 99.4, 99.3,
immunized with the subunit vaccine (Figure 3), highlighting
that the inner core is antigenic when presented in a proper
context.
CONCLUSION
■
The successful preparation of hexasaccharide 1, which is
derived from the inner core of the LPS of F. tularensis, relied on
the use of an orthogonal protected disaccharide that made it
possible to establish the optimal glycosylations sequence to
prepare a highly crowded 1,2,3-cis configured branching point.
In particular, the approach employed a β-D-Man-(1→4)-D-Man
disaccharide modified with the orthogonal protecting groups
diethylisopropylsilyl (DEIPS), 2-naphthylmethyl (Nap), allyl
ether (All), and levulinoyl (Lev) ester. Furthermore, a variety of
methods were exploited to control anomeric selectivities of the
glycosylations including steric, conformational, and solvent
effects and classical and auxiliary mediated neighboring group
participation. These strategic considerations will be important
for the preparation of other highly branched oligosaccharides. It
also highlights that the branched nature of many biologically
important oligosaccharides complicates the development of
routine synthesis procedures based, for example, on automated
1
polymer-supported synthesis. The comparison of the H NMR
spectra of isolated material and the synthetic derivative
confirmed the reported structural assignment of the inner
core oligosaccharide of F. tularensis. The fact that no antigenic
responses to the inner core oligosaccharide were observed in
mice immunized with a whole bacterial vaccine (LVS) indicates
that it may not be suitable for the development of a diagnostic
tool. However, the observation that immunizations with
isolated LPS lead to antibody responses to the inner core
makes it a worthwhile candidate for further exploration as a
vaccine candidate. Future studies will focus on immunizations
with hexasaccharide 1 conjugated to KLH to establish potential
protective properties of this compound.
85.2, 78.5, 76.9, 76.4, 76.0, 76.0, 76.0, 75.8, 75.8, 75.1, 72.9, 72.6, 72.5,
72.5, 71.5, 71.0, 70.9, 70.9, 69.5, 69.4, 69.0, 68.3, 67.9, 66.9, 65.4, 62.6,
61.7, 60.7, 60.6, 60.5, 50.2, 59.6, 50.7, 38.3, 35.0, 26.0, 23.1; HR-
MALDI-TOF/MS (m/z): [M + Na]+ calcd for [C41H72N2O32 + Na]+,
1127.3965; found, 1127.3918.
2-Deoxy-2-amino-α-D-galactosamine-(1→2)-3-O-[α-D-gluco-
pyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-O-[β-D-gluco-
pyranosyl-(1→2)]-α-D-mannopyranosyl-(1→5)-N-biotinyl-3-
amino-propyl-3-deoxy-α-D-manno-octulopyranosidonate (2).
Compound 1 (5.0 mg, 4.5 μmol) was dissolved in PBS buffer pH
7.4 (0.5 mL), and BiotinOSu (1.8 mg, 4.5 μmol) in PBS buffer pH 7.4
(0.2 mL) was added. The resulting mixture was stirred for 3 h at rt.
The mixture was directly transferred to a reverse phase C-18 column
EXPERIMENTAL SECTION
■
General Procedures. 1H and 13C NMR spectra were recorded on
a 300, 500, or a 600 MHz spectrometer. Chemical shifts are reported
in parts per million (ppm) relative to tetramethylsilane (TMS) as the
internal standard. NMR data is presented as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of
doublet, m = multiplet and/or multiple resonances), coupling constant
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Journal of the American Chemical Society
Article
and purified by eluting with 0−10% MeOH/H2O. The appropriate
fractions were collected and concentrated in vacuo to afford 2 (3.7 mg,
62%) as a white solid. 1H NMR (600 MHz, CDCl3): δ 5.51 (d, 1H, J =
3.6 Hz, H-1-D), 5.31 (d, 1H, J = 3.6 Hz, H-1-F), 5.15 (s, 1H, H-1-B),
4.84 (s, 1H, H-1-C), 4.61 (dd, 1H, J = 4.5 Hz, J = 7.8 Hz, CH Biotin),
4.49 (d, 1H, J = 7.8 Hz, H-1-E), 4.61 (dd, 1H, J = 4.2 Hz, J = 8.4 Hz,
CH Biotin), 4.33 (m, 1H, H-2-C), 4.28 (m, 1H, H-2-B), 4.22−4.04
(m, 3H, H-4-A, H-3-B, H-3-C), 3.95−3.22 (m, 29H, H-6a,b-
B,C,D,E,F, H-2-E, H-3-E, H-4-E, H-5-E, H-2-F, H-3-F, H-4-F, H-5-
F, H-3-D, H-4-D, H-5-D, H-4-C, H-5-C, H-4-B, H-5-B, H-5-A, H-6-A,
H-7-A, H-8-A), 3.11 (dd, 1H, J = 10.8 Hz, J = 4.2 Hz, H-2-D), 3.00
(dd, 1H, J = 12.6 Hz, J = 4.8 Hz, CHH Biotin), 2.78 (d, 1H, J = 13.2
Hz, CHH Biotin), 2.24 (t, 1H, J = 7.2 Hz, CH2 Biotin), 2.05 (dd, 1H, J
= 12.6 Hz, J = 4.2 Hz, H-3a-A), 1.79−1.56 (m, 8H, CH2 Linker, 2 x
CH2 Biotin, H-3b-A), 1.42−1.38 (m, 2H, CH2 Biotin); 13C NMR (125
MHz, CDCl3) δ 176.5, 175.0, 165.2, 101.5, 100.1, 99.8, 88.7, 99.2,
97.2, 79.0, 76.7, 76.5, 76.5, 76.2, 75.9, 75.8, 75.1, 72.9, 72.7, 72.6, 72.5,
71.4, 71.2, 71.0, 70.8, 69.6, 69.4, 69.3, 68.3, 68.1, 66.9, 65.5, 63.0, 61.8,
60.9, 60.7, 60.6, 60.4, 60.1, 59.5, 55.1, 53.7, 50.6, 39.5, 36.5, 35.4, 35.0,
28.1, 27.7, 27.5, 25.0; HR-MALDI-TOF/MS (m/z): [M + Na]+ calcd
for [C51H86N4O34S + Na]+, 1353.4741; found, 1353.4726.
2-Deoxy-2-amino-α-D-galactosamine-(1→2)-3-O-[α-D-gluco-
pyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-O-[β-D-gluco-
pyranosyl-(1→2)]-α-D-mannopyranosyl-(1→5)-N-thioacetyla-
cetyl-3-amino-propyl-3-deoxy-α-D-manno-octulopyranosido-
nate (3). Compound 1 (5.0 mg, 4.5 μmol) was dissolved in DMF (0.5
mL), and SAMAOpfp (1.4 mg, 4.5 μmol) in DMF (0.2 mL) and
DIPEA (0.2 uL, 9.0 μmol) were added. The resulting mixture was
stirred for 3 h at rt. The mixture was directly transferred to a reverse
phase C-18 column and purified by eluting with 0 −10% MeOH/H2O.
The appropriate fractions were collected and concentrated in vacuo to
afford 3 (2.9 mg, 53%) as a white solid. 1H NMR (600 MHz, D2O): δ
5.34 (s, 1H, H-1-D), 5.09 (d, 1H, J = 3.6 Hz, H-1-F), 4.90 (s, 1H, H-1-
B), 4.60 (s, 1H, H-1-C), 4.27 (d, 1H, J = 7.8 Hz, H-1-E), 4.12−4.14
(m, 1H, H-2-C), 4.05−4.00 (m, 1H, H-2-B), 3.97 (t, 1H, J = 9.0 Hz,
H-5-D), 3.88−3.86 (m, 1H, H-4-A), 4.10−3.00 (m, H, H-6a,b-
B,C,D,E,F, H-3D, H-4-D, H-2-F, H-3-F, H-4-F, H-5-F, H-3-B, H-4-B,
H-3-C, H-4-C, H-5-C, H-3-E, H-4-E, H-5-E, CH2 Linker, H-2-E, H-2-
D, H-3-E, H-5-B, CH2 Linker), 1.92−1.87 (m, 1H, H-3a-A), 1.56−
1.46 (m, 3H, CH2 Linker, H-3b-A). HR-MALDI-TOF/MS (m/z): [M
+ Na]+ calcd for [C45H76N2O34S + Na]+, 1243.3897; found,
1243.3847.
Conjugation of 3 to Keyhole Limpet Hemocyanin (KLH).
Compound 3 (1.5 mg, 1.3 μmol) was dissolved in DMF (0.5 mL), and
5% ammonia in DMF (50 μL) was added. After 2 h, MALDI-TOF
showed complete removal of the S-acetyl, and the mixture was
concentrated in vacuo. The residue was dissolved in PBS buffer pH 7.2
(1.0 mL), and a solution of maleimide-activated mcKLH (4 mg in 0.5
mL water) was added. The resulting mixture was stirred for 2 h at rt.
The mixture was purified by spin filtration. Analysis of the KLH
conjugate using high-performance anion-exchange chromatography
(HPAEC) showed that on average, 339 hexasaccharides were
conjugated per KLH molecule (see Supporting Information). Since
one KLH molecule has 522 maleimide molecules this corresponds to a
conversion of 65%.
Dose and Immunization Schedule. Specific-pathogen-free,
female BALB/c mice were purchased from Charles Rivers Laboratories
(St. Constant, Que.). Mice were maintained and used in accordance
with the recommendations of the Canadian Council on Animal Care
Guide to the Care and Use of Experimental Animals and entered the
experiments between 7 and 10 weeks of age. For LVS immunization,
mice were immunized p.o. on day 0 and 14 with 2 × 108 CFU F.
tularensis LVS (actual confirmed inocula: 1.6 × 108/mouse for the first
immunization and 2 × 108/mouse for the second immunization) or
PBS as control as described previously.32 The mice were killed on day
35 for serum collection. The serum samples from the mice immunized
with a vaccine consisting of the O-polysaccharide of the F. tularensis
chemically lipopolysaccharide (LPS) conjugated to bovine serum
albumin (BSA-O-PS conjugate) were kindly provided by Dr. Wayne
Conlan (National Research Council Canada, Ottawa, Canada). The
preparation of the glycoconjugate vaccine was described previously in
details.6 Mice were immunized subcutaneously at 0, 28, and 56 days
with 20 μg of the glycoconjugate emulsified in a 1:3 ratio with
incomplete Freud’s adjuvant in a total volume of 0.1 mL. Mice were
killed on day 70 for serum collection.
Serologic Assay. IgG antibody titers against the inner core of F.
tularensis LPS were determined by enzyme-linked immunosorbent
assay (ELISA). Reacti-bind NeutrAvidin-coated and preblocked plates
(Thermo Scientific) were incubated with compound 2 (a stock
solution in DMSO (2 mM) was diluted to 5 μM; 100 μL/well) for 2 h.
Next, serial dilutions of the sera were allowed to bind to immobilized
compound for 2 h. Detection was accomplished by the addition of
alkaline phosphatase-conjugated antimouse IgG (Jackson ImmunoR-
esearch Laboratories Inc.). After addition of p-nitrophenyl phosphate
(Sigma), the absorbance was measured at 405 nm with wavelength
correction set at 490 nm using a microplate reader (BMG Labtech).
The antibody titer was determined by linear regression analysis,
plotting dilution vs. absorbance. Titers were defined as the highest
dilution yielding an optical density of 0.1 or greater over that of
control mouse sera. Experiments were performed in triplicate.
ASSOCIATED CONTENT
* Supporting Information
■
S
The preparation of the starting materials, assembly of the
oligosaccharides, and copies of NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
gjboons@ccrc.uga.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. John Glushka for assisting with NMR
experiments and Dr. Wayne Conlan for providing serum
samples of LPS-immunized mice. Research reported in this
publication was supported by the National Institute of General
Medical Sciences of the National Institutes of Health
(R01GM065248, G.-J.B.).
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