Chemical synthesis of all phosphatidylinositol mannoside (PIM) glycans from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/ja806283e

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (Pl) and phosphatidylinositol mono- to hexa-mannoside (PIM₁ to PIM₆). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyholelimpet hemocyanin (KLH).

Full text of DOI:10.1021/ja806283e

Published on Web 11/17/2008
Chemical Synthesis of All Phosphatidylinositol Mannoside
(PIM) Glycans from Mycobacterium tuberculosis
Siwarutt Boonyarattanakalin,†,‡ Xinyu Liu,†,§ Mario Michieletti,†,| Bernd Lepenies,†
and Peter H. Seeberger*,†
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH) Zu¨rich,
Wolfgang-Pauli-Str. 10, HCI F312, 8093 Zu¨rich, Switzerland, and Sirindhorn International
Institute of Technology, Thammasat UniVersity, P.O. Box 22 Thammasat-Rangsit Post Office,
Pathumthani 12121, Thailand
Received August 8, 2008; E-mail: seeberger@org.chem.ethz.ch
Abstract: The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis
vaccine to protect humans against TB have prompted investigations into alternative approaches to combat
this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs)
are biologically important glycoconjugates and represent common essential precursors of more complex
mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan
capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate
the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the
function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient
synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside
(PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as
well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker
for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic
PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific
intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune
stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-
limpet hemocyanin (KLH).
multidrug-resistant TB9 and the low efficacy of the BCG
vaccine. Therefore, the exploration of novel drug targets and
Introduction
Tuberculosis (TB) is a complex disease and a major cause
of mortality worldwide.1-3 Despite the development of new
treatments, TB remains a global health concern.4,5 Annually,
there are more than seven million new cases and two million
deaths caused by TB.6 Coinfection with HIV leads to an
exacerbation of the disease4 and contributes to higher mortality
in HIV patients.6,7 Programs to combat TB in many countries
have failed to eradicate TB,8 partly due to the spread of
vaccines against Mycobacterium tuberculosis (Mtb), the main
causative pathogen of TB, is essential.
Among pathogenic bacteria, Mtb causes more deaths in
humans than any other pathogen.6,10,11 Approximately one-third
of the world population has already been infected by Mtb.4 Mtb
is an intracellular pathogen that has evolved to persist efficiently
in infected macrophages.4,8,12 The composition of the Mtb cell
wall is important for the interaction with host cells during the
initial steps of the infection. Later, cell wall components play
a crucial role in modulating the pro-inflammatory response by
macrophages and also serve as a protective barrier to prevent
antituberculosis agents from permeating inside. Consequently,
the antibiotics used for the treatment of tuberculosis require
long-term administration.5 Mortality in people living in develop-
ing countries is high since their access to these antibiotics is
often limited and compliance with treatment courses is low.
The major components of the mycobacterial cell wall are the
mycoyl arabinogalactan-peptidoglycan (mAGP) complex and
interspersed glycolipids including ManLAM, LAM, LM, and
† Swiss Federal Institute of Technology (ETH) Zu¨rich.
‡ Sirindhorn International Institute of Technology.
§ Current address: Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
MA 02115.
| Visiting Ph.D. student from Universita` degli Studi del Piemonte
Orientale “Amedeo Avogadro”, Novara, Italy.
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2, 189–202.
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A R T I C L E S
Boonyarattanakalin et al.
of PIMs was emphasized by the finding that PIMs bind to
receptors on both phagocytic17,24,25 and nonphagocytic12 mam-
malian cells. Recently, it has been shown that PIMs, but neither
LAM nor ManLAM interact with the VLA-5 on CD4+
T
lymphocytes and induce the activation of this integrin.22 These
findings suggest that PIMs are not only secreted to the
extracellular environment, but also exposed on the surface of
Mtb to interact with host cells.
Although different functions have been ascribed to the PIMs,
it remains to be determined whether and to which extent the
different PIM substructures display biological activity. A better
understanding of the mycobacterial cell wall biosynthesis is
required to be able to counteract with the problems of drug
resistance and bacterial persistence. Synthetic PIMs represent
important biochemical tools to elucidate biosynthetic pathways
and to reveal interactions with receptors on host cells. PIMs
are potential vaccine antigens and/or adjuvants.
Several synthetic PIMs containing fewer mannoside units
have been synthesized employing various chemical methodolo-
gies.26-33 In contrast to PIM3 and PIM4 that contain only R-1,6
mannosidic linkages, PIM5 and PIM6 also incorporate R-1,2
mannosides that might contribute to different biological activities
of these PIMs. None of the studies to date utilized synthetic
PIMs that contain linkers for immobilization. Coupling of
synthetic PIMs to carrier proteins, beads, quantum dots, mi-
croarray or surface plasmon resonance (SPR) surfaces opens a
host of options for biochemical studies. Here, we report the
efficient synthesis of the carbohydrate portion of all PIMs
including phosphatidylinositol (PI) and PIM1 to PIM6 (Figure
2). The native diacylglycerol phosphate at the C-1 position of
myo-inositol is replaced by a 6-thiohexyl phosphate residue for
immobilization of the synthetic PIMs on surfaces.
Figure 1. Structural features of PIMs, LM, LAM, and ManLAM of
Mycobacterium tuberculosis. PIMs are the common precursors of more
complex components of the mycobacterial cell wall including lipomannan
(LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan
(ManLAM). (a, b, and c are varied; typically, R2 is tuberculostearic acid,
R1 and R3 are various fatty acids.)
PIMs. While the mAGP complex is covalently attached to the
bacterial plasma membrane, the glycolipids are noncovalently
attached through their phosphatidyl-myo-inositol (PI) anchor.13-15
PIMs constitute the only conserved substructure of LM, LAM,
and ManLAM (Figure 1). The inositol residue of PI is
mannosylated at the C-2 position to form PIM1 and further at
the C-6 position to form PIM2, one of the two most abundant
naturally occurring PIMs, along with PIM6. Further R-1,6
mannosylations give rise to PIM3 and PIM4sthe common
biosynthetic precursors for PIM5, PIM6, and the much larger
LMstructures.LAMisconstitutedbyattachmentofarabinanssthe
repeating units of R-1,5 arabinose terminated with a single ꢀ-1,2
arabinose to mannose units of LM. The nonreducing end
arabinose in the arabinan moiety of LAM can be capped at the
C-5 position with one or two R-mannose units to furnish
ManLAM.
Results and Discussion
Retrosynthetic Analysis. The overall structure of the synthetic
PIM targets (Figure 2) can be attained by the convergent union
of oligomannosides with D-myo-inositol containing pseudosac-
charides and a thiol-terminated phosphate linker (Scheme 1).
Late-stage couplings between protected oligosaccharide frag-
ments (1-4) and 8 allow for parallel syntheses of the intermedi-
ates for all target molecules. The key glycosylations in these
syntheses are the couplings between mannosyl phosphate 1,
oligomannosyl trichloroacetimidates (2-4) and the common
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Infect. Immun. 2001, 69, 2172–2179.
Among the surface components involved in the Mtb interac-
tion with host cells, PIMs play a crucial role in the modulation
of the host immune response.16-23 The functional importance
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2004, 3292–3303.
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Chemical Synthesis of All PIM Glycans
A R T I C L E S
Figure 2. Structures of synthetic PI and PIM1 to PIM6.
Scheme 1. Retrosynthetic Analysis for the Assembly of Synthetic PIMs
pseudotrisaccharide 8. The two main carbohydrate moieties are
coupled, followed by protecting group manipulations. Subse-
quently, a phosphate diester linker is installed using an H-
phosphonate followed by oxidation of phosphorus. Since the
target molecules contain sulfur that is known to deactivate the
Pd/C catalyst, the permanent benzyl protecting groups are
globally removed via Birch reduction.
The stereoselectivity of each glycosydic bond formation is
ensured by neighboring C-2 acyl participating groups. In this study,
we employed an anomeric dibutyl phosphate ester as a leaving
group for the mannose building blocks that can be readily prepared.
This method proved advantageous when compared to previous PIM
syntheses. Three mannose building blocks (1, 5, and 10) are needed
in addition to the inositol building block.
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A R T I C L E S
Boonyarattanakalin et al.
Scheme 2. Efficient Multi-Gram Preparations of Mannose Building
Scheme 4. Assembly of myo-Inositol Containing
Blocks via Bicyclic and Tricyclic Orthoester Intermediatesa
Pseudotrisaccharide 8a
a Reagents and conditions: (a) i. Ac2O, HClO4 (cat.), ii. HBr/HOAc, iii.
MeOH, Lutidine, 90%, three steps; (b) i. NaOMe/MeOH/THF, ii. NaH,
BnBr, DMF, quant. two steps; (c) HOP(O)(OBu)2, 4 Å MS 93%; (d) ref 35
- i. BzCl, Py, ii. HBr/HOAc, iii. AllOH, Lutidine, iv. NaOMe/MeOH/THF,
reflux, v. CSA, MeCN, vi. NaH, BnBr, DMF, 70%, six steps; (e)
HOP(O)(OBu)2, 4 Å MS, 97%; (f) TIPSCl, NEt3, DMAP, CH2Cl2, 91%;
(g) AllOH, BF3-Et2O, CH2Cl2, 99%.
a Reagents and conditions: (a) TMSOTf, Toluene, -40 °C, 90%; (b)
AcCl, MeOH, CH2Cl2, 0 °C, quant.; (c) LevOH, DIPC, DMAP, quant.; (d)
DDQ, CH2Cl2, MeOH, 0 °C, 95%; (e) 1, TBDMSOTf, Toluene, -40 °C,
95%, (see Table 1); (f) H2NNH3OAc, MeOH, rt, 89%.
Scheme 3. Modified Synthesis of
Table 1. Effects of Promoter and Temperature on the
Glycosylation of Glycosyl Phosphate 1 and myo-inositol
Intermediate 20
1-O-Acetyl-3,4,5-tri-O-benzyl-myo-inositol (16)a
entry
promoter
temperature (°C)
yield (%)
1
2
3
4
5
TMSOTf
-40
-40
-10
rt
15
27
57
55
95
TBDMSOTf
TBDMSOTf
TBDMSOTf
TBDMSOTf
a Reagents and conditions: (a) i. Imidazole, TIPSCl, DMF 0 °C to rt, ii.
NaH, BnBr, DMF, 0 °C to rt, iii. TBAF, THF, 99%, three steps; (b) i.
SO3-Py, DIPEA, DMSO, CH2Cl2, 0 °C to rt, ii. K2CO3, Ac2O, MeCN,
reflux, iii. Hg(CF3COO)2, Acetone/H2O (4:1), rt, 1 h, then NaOAc (aq),
NaCl (aq), 0 °C to rt, iv. NaBH(CH3COO)3, AcOH, MeCN, 0 °C to rt,
40%, four steps.
0
groups were introduced at C1 and C2 of the D-myo-inositol
respectively as previously described37 to furnish 11, ready for
further decoration at the C6 hydroxyl group.
Syntheses of Monosaccharide Building Blocks. Mannosyl
building blocks 1, 5, and 10 were synthesized from mannose
bicyclic and tricyclic orthoesters (6, 12, Scheme 2).34,35 Starting
from D-mannose, mannosyl phosphate 1 was accessed in six
steps by dibutyl phosphoric acid opening of the bicyclic
orthoester 7. Mannosyl tricyclic orthoester 6 is readily available
from D-mannose over six high yielding steps.35 This process
required only one purification at the last step and gave 6 in
70% overall yield. The versatile intermediate 6 was opened by
allyl alcohol upon activation with BF3 ·Et2O to afford 5 in
excellent yield. Treatment of orthoester 6 with dibutyl phosphate
selectively opened the tricyclic orthoester to furnish glycosyl
phosphate 13, leaving the C-6 hydroxyl group unprotected. The
installation of a triisopropylsilyl (TIPS) group was straightfor-
ward and furnished building block 10.
The previously reported synthetic route to the differentially
protected myo-inositol by Fraser-Reid et al.36 was modified
(Scheme 3). Methyl glucopyranose was quantitatively converted
to 15 in three consecutive steps. A Parikh-Doering reaction
oxidized the primary hydroxyl group in 15 to an aldehyde in
quantitative yield. Using this oxidation, we avoided complica-
tions arising from the urea byproduct created when dicyclo-
hexylcarbodiimide (DCC) was used as activator. The sulfate
byproduct was readily removed by water extraction. The
partially protected myo-inositol 16 was prepared from 15 in 40%
yield over four consecutive steps. The allyl and NAP protecting
Assembly of myo-Inositol Containing Pseudosaccharides. The
myo-inositol containing pseudotrisaccharide 8 was assembled
in a stepwise manner. Glycosylation of inositol 11 with
mannosyl phosphate 10 that contained a C6-TIPS ether as a
temporary protecting group was found to be optimal at
-40 °C, in toluene, and promoted by a stoichiometric amount
of TMSOTf (Scheme 4). Under these conditions the reaction
gave a good yield with complete R-selectivity. To sustain further
glycosylations, the temporary TIPS protecting group was
replaced by the levulinoyl (Lev) group. The presence of TIPS
rather than Lev on the C6 hydroxyl group of 10 was found
necessary to balance its reactivity with inositol 10 to obtain high
yield and selectivity, as observed in a previous study.37
Treatment of 19 with DDQ unmasked the C2 hydroxyl group
on inositol to give 20 that served in turn as nucleophile during
the next mannosylation.
The second mannosylation on the C2 hydroxyl group of
pseudodisaccharide 20 was found to be nontrivial (Table 1).
Activation by TMSOTf afforded the desired pseudodisaccharide
21 in just 15% yield (Table 1, Entry 1). Decomposition of 1 to
form the anomeric alcohol was observed instead. Switching the
promoter from TMSOTf to the milder activator TBDMSOTf
dramatically improved the yield of the desired product (Table
1, Entry 2). This observation suggested a possible reactivity
mismatch between highly activated 1 and less activated 20. The
glycosylation was thus improved by reducing the reactivity of
1 with TBDMSOTf. The activity of the less reactive 20 was
increased by higher reaction temperatures. Product 21 was
(34) Ravida, A.; Liu, X.; Kovacs, L.; Seeberger, P. H. Org. Lett. 2006, 8,
1815–1818.
(35) Liu, X.; Wada, R.; Boonyarattanakalin, S.; Castagner, B.; Seeberger,
P. H. Chem. Commun. 2008, 3510–3512.
(36) Jia, Z. J.; Olsson, L.; Fraser-Reid, B. J. Chem. Soc., Perkin Trans. 1
1998, 631.
(37) Liu, X.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006,
128, 3638–3648.
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Chemical Synthesis of All PIM Glycans
A R T I C L E S
Scheme 5. Protecting Group Manipulations on myo-Inositol 16 for
PI Intermediate 23 and PIM1 Intermediate 24a
at -10 °C. After quenching with triethylamine, the concentrated
crude products were directly converted to obtain the benzylated
products. When the larger structure 4 was used for glycosylation,
the coupling became more sluggish and resulted in the hydroly-
sis of 4. A higher temperature (0 °C) was needed to obtain the
4 + 3 glycosylation product 46 (see Experimental Section).
Pseudoheptasaccharide 46 was the largest oligosaccharide
assembled in this series and consisted of fragments of all smaller
oligosaccharides. Thus, 46 was analyzed extensively by C-H
coupled HSQC to confirm its structural identity. 2D-NMR data
elucidated six anomeric proton signals with typical38 R-manno
JC1,H1 couplings.
a Reagents and conditions: (a) Ac2O, DMAP, Py, 70%; (b) NaH, BnBr,
DMF, 0 °C to rt, quant.
Removal of O-Allyl Protecting Group on Inositol. Protocols
to cleave the C-1 O-allyl group on inositol attached to
oligosaccharides, performed by using PdCl2, have been reported
to give moderate yields.32,40-43 This literature precedence was
reflected in our study as well. Different methods to remove the
O-allyl group were explored on substrate 31 (Scheme 7 and
Table 3). The hydrogen activated iridium complex
Ir{(COD)[PH3(C6H5)2 ]2 }PF6 was found to be the most efficient
reagent to isomerize the allyl group to the corresponding enol
ether. In the same pot, a catalytic amount of p-toluenesulfonic
acid (p-TsOH) was added to cleave the enol ether and liberate
the C1 hydroxyl of pseudodisaccharide 38 in quantitative yield.
This two step procedure was applied to the larger oligosaccha-
rides 32 to 36 as well. However, while the isomerizations
mediated by the iridium complex worked smoothly, an excess
of p-TsOH (10 equiv) was required to cleave the enol ether
and furnish 39 - 43 (Scheme 7, entry 3).
Phosphorylation and Global Deprotection. The phosphate
moiety accompanied by a terminal thiol linker was installed on
the inositol C1 hydroxyl group of the oligosaccharide backbone
37-43 using a H-phosphonate (Scheme 8). Substrates 37-43
were treated with pivaloyl chloride in the presence of linker 44
and pyridine. Subsequently, in the same pot, the H-phosphonate
diesters were oxidized with iodine and water to provide the fully
benzylated phosphodiesters 45 as triethylamine salts in excellent
yield. Global removal of benzyl protecting groups of analogs
45a-g was achieved under Birch reduction conditions. The fully
protected compounds were treated with sodium dissolved in
ammonia to furnish the final products PI and PIM1-PIM6
(Figure 2). Small amounts of incompletely reduced products
were observed containing some remaining benzyl groups. These
side products were separated by extraction with chloroform and
converted to the final products by resubmission to Birch
reduction. The final products were formed as a mixture of
monomers and disulfide dimers. Treatment with one equivalent
of tris(carboxyethyl) phosphine hydrochloride (TCEP) im-
mediately prior to conjugation of the final compounds ensured
that PI and PIM1-PIM6 were present as monomers.
obtained in excellent yield (95%) and selectivity by performing
the glycosylation at 0 °C (Table 1, Entry 5). The R linkages in
21 were confirmed by 2D NMR. 1H-13C coupled HSQC NMR
1
indicated H1-13C1 coupling constants (JC1,H1) of 178 Hz at
the anomeric position of the mannose connected to the C2 of
inositol and 182 Hz at the anomeric position of the mannose
on C6 of inositol. JC1,H1 of ꢀ mannosidic linkages are typically
lower at around 159 Hz.38
Removal of the Lev group in 21 was achieved by treatment
with hydrazine acetate in methanol and required careful
monitoring. Longer reaction times resulted predominantly in the
reduction of the allyl moiety to a propyl group. Partially
protected inositol 16 was subjected to protecting group ma-
nipulations to furnish the inositol intermediates for PI and PIM1
(Scheme 5). Based on reactivity differences, the equatorial C6
hydroxyl group of the diol 22 was selectively acetylated to afford
23 as the intermediate en route to PIM1. The PI intermediate
24 was obtained in parallel by benzylation of the common
intermediate 22.
Assembly of Oligomannoside Fragments. The oligomannoside
trichloroacetimidates 2, 3, and 4 were assembled in linear
fashion (Scheme 6). All glycosylations employed mannosyl
phosphate 1 and TMSOTf as activator. The R-1,6 glycosydic
bond was readily formed at 0 °C in quantitative yield. A lower
temperature (-40 °C) was required to efficiently install 1,2
glycosylic linkages with complete R-selectivity. Deallylation of
25-27 was performed by allylic substitutions mediated by a
palladium complex to yield the corresponding anomeric alcohols
28-30. Finally, conversion to the glycosyl trichloroacetimidates
2-4 was carried out using sodium hydride as base.
Assembly of Protected PIM Backbones. Prior to phosphory-
lation, all protected PIM oligosaccharide backbones were
obtained by late-state glycosylations (Table 2). Following these
glycosylations all ester protecting groups were removed with
sodium methoxide in methanol at elevated temperature before
masking the free hydroxyl groups as benzyl ethers. These
protecting group manipulations were performed to avoid the
persistence of O-benzoate protecting groups under Birch condi-
tions in the final deprotection.39
Coupling between mannosyl phosphate 1 and inositol 23 gave
pseudodisaccharide 31, the backbone of PIM1. To access the
PIM2 backbone, pseudotrisaccharide fragment 8 was directly
used as the starting material to be transformed into backbone
32. The glycosylation products from couplings (Table 2, entry
3-5) between the oligomannosyl trichloroacetimidates (1-3)
and the common pseudotrisaccharide 8 were cleanly achieved
PIM Microarrays to Determine Binding to DC-SIGN. To
study the interactions of synthetic PI and PIMs with the protein
DC-SIGN on a microarray, PI and PIMs were immobilized on
a maleimide activated glass slide via their thiol handle following
established protocols (Figure 3).44 DC-SIGN is an important
receptor on dendritic cells and contributes to the initiation of a
pro-inflammatory response by host cells.45-47 One of the
functions of DC-SIGN is the recognition of evolutionary
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Boonyarattanakalin et al.
a
Scheme 6. Assembly of Oligomannosylating Reagents 2-4 for the Synthesis of PIM4-PIM6
a Reagents and conditions: (a) TMSOTf, CH2Cl2, -10 °C, quant.; (b) AcCl, MeOH, CH2Cl2, 0 °C, 91%; (c) 1, TMSOTf, -40 °C, Toluene, 95%: (d)
AcCl, MeOH, CH2Cl2, 0 °C, 84%; (e) 1, TMSOTf, Toluene, -40 °C, 96%; (f) Pd(OAc)2, MeOH, PPh3, Et2NH, 77% for 28, 95% for 29, and 83% for 30;
(g) Cl3CCN, NaH, rt, 85% for 2, 86% for 3, and 89% for 4.
conserved pathogenic structures that are secreted or exposed
on the surface of viruses or bacteria.46,48-50 Upon binding to
DC-SIGN, the antigens are internalized, processed and later
presented on the surface of dendritic cells together with
costimulatory molecules.51,52 Mycobacteria also use DC-SIGN
as a receptor to enter dendritic cells.51
four C57BL/6 mice per group were prime-boost immunized with
the model antigen keyhole-limpet hemocyanin (KLH) covalently
linked to PIM6. As expected, immunization with the pure
antigen KLH resulted in detectable anti-KLH antibody levels.
Antibody production in the presence of the well-established
adjuvants Freund’s adjuvant, alum and CpG, increased sub-
stantially. In comparison, conjugation of PIM6 glycan to KLH
also resulted in a marked increase of anti-KLH antibodies that
was statistically significant for each serum dilution compared
to KLH alone (Figure 4A). To address the mechanism causing
the increased antibody production after covalent attachment of
PIM6 to KLH, we restimulated spleen cells of immunized mice
with KLH ex ViVo and measured proliferation. Spleen cell
proliferation of mice that had been immunized with KLH-PIM6
was significantly increased indicating that T cell priming was
stimulated by PIM6 glycan (Figure 4B). It is also known that
adjuvant properties not only depend on antibody production and
T cell proliferation, but also on other T effector functions such
as cytokine production. To this end, we measured IFN-γ
production of T cells by ELISpot analysis. The frequency of
IFN-γ producing T cells in spleen was determined upon
restimulation of T cells with KLH. The ability of T cells to
produce IFN-γ was increased in spleen cells of mice that had
been immunized with KLH-PIM6 conjugate (Figure 4C). The
effect was even stronger than with the well-established adjuvants
Freund’s adjuvant, alum or CpG, which highlights the immu-
nostimulatory capacity of synthetic PIM6 glycan. Concanavalin
A was used as a positive control since it serves as a T cell
mitogen and stimulates all T cells to the same extent.
Glass slides printed with the immobilized PI and
PIM1-PIM6 were incubated with a DC-SIGN solution in buffer
at room temperature to allow DC-SIGN to bind to the im-
mobilized PIMs. Excess DC-SIGN was washed off and bound
DC-SIGN was detected by incubation with a fluorescein-
conjugated anti-DC-SIGN antibody. The difference in DC-SIGN
binding affinity to the synthetic PIM compounds was assessed
semiquantitatively by monitoring the fluorescence intensity via
a fluorescence scanner (for fluorescent intensity data, see
Supporting Information). Synthetic PIMs bind to DC-SIGN in
a specific manner (Figure 3). Although both synthetic analogs
of the most abundant PIM2 and PIM6 are recognized by DC-
SIGN, the larger synthetic oligosaccharides PIM5 and PIM6
bound to DC-SIGN to a greater extent. This observation
underlines the significance of the R-1,2- mannose motif present
in both PIMs and ManLAM structures.53
Adjuvant Activity of PIMs. An important feature of natural
PIMs is their ability to induce a host cell immune response. To
investigate immunostimulatory effects of these synthetic PIMs
(44) de Paz, J. L.; Horlacher, T.; Seeberger, P. H. Methods Enzymol. 2006,
415, 269–292.
(45) van Kooyk, Y.; Geijtenbeek, T. B. Nat. ReV. Immunol. 2003, 3, 697–
709.
Recognition of PIM6 by pattern recognition receptors on
antigen-presenting cells might provide a danger signal, thereby
facilitating enhanced uptake of the model antigen and increased
expression of costimulatory molecules. The effect of PIM6 on
T cell proliferation and T cell effector functions such as IFN-γ
production clearly indicates that antigen presentation by APCs
and T cell activation are increased by PIM6 glycan.
The synthetic PI and PIM5-PIM6 described here will be
suitable for conjugation with other appropriate surfaces such as
fluorescent nanocrystals, beads or fluorophores to generate probes
for cellular assays. Such tools may shed light on the mechanism
(46) Cambi, A.; Figdor, C. G. Curr. Opin. Cell Biol. 2003, 15, 539–546.
(47) Banchereau, J.; Steinman, R. M. Nature 1998, 392, 245–252.
(48) Cambi, A.; Koopman, M.; Figdor, C. G. Cell. Microbiol. 2005, 7,
481–488.
(49) Su, S. V.; Hong, P.; Baik, S.; Negrete, O. A.; Gurney, K. B.; Lee, B.
J. Biol. Chem. 2004, 279, 19122–19132.
(50) Geijtenbeek, T. B.; Engering, A.; Van Kooyk, Y. J. Leukoc. Biol. 2002,
71, 921–931.
(51) De Libero, G.; Mori, L. Nat. ReV. Immunol. 2005, 5, 485–496.
(52) De Libero, G.; Mori, L. FEBS Lett. 2006, 580, 5580–5587.
(53) Koppel, E. A.; Ludwig, I. S.; Hernandez, M. S.; Lowary, T. L.;
Gadikota, R. R.; Tuzikov, A. B.; Vandenbroucke-Grauls, C. M.; van
Kooyk, Y.; Appelmelk, B. J.; Geijtenbeek, T. B. Immunobiology 2004,
209, 117–127.
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Chemical Synthesis of All PIM Glycans
A R T I C L E S
Table 2. Assembly of Fully Protected PIM1-PIM6 Backbones: Union of (oligo)Mannosyl Fragment (X) and Inositiol-Containing
Pseudosaccharide Fragment (Y)
by which PIM structures on Mtb can influence bacterial trafficking
in host cells. The synthetic compounds can also be attached to
affinity columns in search for proteins or enzymes in cell lysates
that interact with PIMs. Moreover, the synthetic PIMs can be used
as substrates to explore biosynthetic pathways of the PIMs.
We are investigating the possibility of applying synthetic PIMs
as antigens to elicit an immune response against Mtb as well as
their adjuvant properties in ViVo. For these purposes, the synthetic
compounds can be conjugated to different model antigens.
robust and practical synthesis to the PIM molecules was
developed utilizing mannosyl bicyclic and tricyclic orthoesters
and mannosyl phosphates. The key intermediate orthoesters
allowed for rapid and scalable syntheses of mannoside building
blocks and the glycosylations of the mannosyl phosphates
resulted in excellent yields and stereoselectivity. All synthetic
PIMs are equipped with a thiol linker to be readily immobilized
on microarray surfaces. Thus, the synthetic PIMs represent tools
for various biological studies. An application of the synthetic
PI and PIMs for interaction with the protein DC-SIGN was
Conclusion
In this study, the efficient synthesis of all PIMs including
phosphatidylinositol (PI) and PIM1 to PIM6 was reported. A
(54) Takahashi, H.; Kittaka, H.; Ikegami, S. J. Org. Chem. 2001, 66, 2705–
2716.
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A R T I C L E S
Boonyarattanakalin et al.
Scheme 7. Removal of Allyl Protecting Groups on C1 myo-Inositol of Fully Protected PI and PIM2-PIM6
Table 3. Removal of Allyl Protecting Group on
Pseudo-Disaccharide 31
protein concentration in the eluate was determined by measuring
the absorption at a wavelength of 280 nm.
Female C57BL/6 mice (6-8 weeks old) were housed in the HCI
rodent center, ETH Zu¨rich, and were provided food and water ad
libitum. On day 0 four mice per group were s.c. immunized with
KLH alone (group 1), KLH in complete Freund’s adjuvant (group
2), KLH with alum (group 3), KLH with CpG (group 4) or KLH
coupled to PIM6 (group 5). On day 10, mice received a boost
immunization with KLH alone (group 1), KLH in incomplete
Freund’s adjuvant (group 2), KLH with alum (group 3), KLH with
CpG (group 4) or KLH coupled to PIM6 (group 5). The amount of
KLH was adjusted to 50 µg per mouse and immunization. On day
entry
conditions
yield
1
tBuOK, DMSO, 80 EC, then I2,
THF/H2O TMSOTf
Pd(OAc)2, PPh3, HNEt2,
10%,
(decompostion)
no reaction
2
3
4
CH2Cl2/MeOH (2:1)
[Ir(COD)(PCH3Ph2)2]PF6 (cat.), H2,
THF then I2 in THF/H2O (2:1)
[Ir(COD)(PCH3Ph2)2]PF6 (cat.), H2,
THF then p-TsOH (cat.) in DCM/MeOH (1:3)
30%
quantitative
Scheme 8. Phosphorylation of Oligosaccharides 37-43 and Global
Deprotection under Birch Reduction Conditionsa
a Reagents and conditions: (a) i. 44, PivCl, pyridine, ii. I2, H2O, pyridine,
90% to quant., 2 steps; (b) i. Na/NH3 (l) /t-BuOH, -78 °C, ii. MeOH, 65%
for PI, 43% for PIM1, 56% for PIM2, 91% for PIM3, 65% for PIM4, 88%
for PIM5, and 84% for PIM6.
demonstrated. The difference in DC-SIGN binding affinity
among synthetic PI and PIM compounds was observed in a
specific manner. Immunization experiments in mice revealed
the potential of synthetic PIMs to serve as immune stimulators.
Experimental Section
Immunization of Mice and Detection of anti-KLH
Antibody Levels in Sera. Preparation of keyhole limpet hemocya-
nin (KLH) in complete/incomplete Freund’s adjuvant was per-
formed by mixing KLH with Freund’s adjuvant in a 1:1 volume
ratio. For coupling of PIM6 to KLH, PIM6 was incubated with
Tris(2-carboxyethyl)phosphine HCl (TCEP) in equal molar ratio
for one hour at rt. A molar excess of PIM6 was then coupled to
KLH using the Imject Maleimide Activated mcKLH Kit (Pierce,
Rockford, IL) according to manufacturer’s instructions. PIM6-KLH
conjugate was purified by gel filtration chromatography and the
Figure 3. Fluorescent scanning of PIM microarray incubated with DC-
SIGN and susequently with fluorescein conjugated antihuman DC-SIGN
antibody (1 h). A PI and PIM immobilized glass slide was incubated with
a solution of DC-SIGN (1 µg/100 µL) in HEPES buffer containing 1%
BSA, 20 mM CaCl2, and 0.5% Tween-20 at room temperature for 1 h. The
slide was washed thoroughly and incubated with a solution of fluorescein
conjugated antihuman DC-SIGN antibody (0.5 µg/100 µL) in HEPES buffer
containing 1% BSA and 0.5% Tween-20 at room temperature for 1 h. The
slide was washed thoroughly and scanned by a fluorescent microarray
scanner. (PBS ) Phosphate-buffered saline, Gal ) Galactose)
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Chemical Synthesis of All PIM Glycans
A R T I C L E S
Figure 4. Immunization studies in mice with the model antigen KLH coupled to PIM6. On day 0, four C57BL/6 mice per group (6-8 weeks) were s.c.
immunized with KLH alone, KLH in complete Freund’s adjuvant, KLH with alum, KLH with CpG or KLH covalently linked to PIM6. On day 10, mice
received a boost immunization with KLH alone, KLH in incomplete Freund’s adjuvant, KLH with alum, KLH with CpG, or KLH-PIM6. (A) On day 17
post immunization blood was taken from the saphenous vein of the immunized mice and levels of anti-KLH antibodies (sum of IgA, IgG and IgM) were
measured by ELISA in serial dilutions of the sera (1:1000, 1:2000, 1:10000, 1:50000, duplicates for each mouse). Data are presented as mean ( SEM for
each group of mice. Statistical analysis was performed with Student’s t test (*, p < 0.05). (B) On day 20 post immunization, 2 × 105 splenocytes were
restimulated with KLH (10 µg/ml) for 24 h and cell proliferation was measured. The results are expressed as a stimulation index (SI) which is the net
proliferation of spleen cell cultures stimulated with 10 µg/ml KLH divided by the net proliferation of spleen cell cultures in medium. Data are presented as
mean ( SEM for each group of mice. Statistical analysis was performed with Student’s t test (*, p < 0.05). The dashed line represents proliferation of spleen
cells from unimmunized mice. (C) On day 20, 2 × 105 splenocytes were stimulated with KLH (10 µg/ml) or Concanavalin A (ConA, 10 µg/ml) in a 96-Well
plate coated with antimouse-IFN-γ and the frequency of IFN-γ producing cells was determined by ELISpot analysis. The results are expressed as spot
forming units (sfu) which is the number of cells producing IFN-γ in each well. Data are presented as mean ( SEM for each group of mice. Statistical
analysis was performed with Student’s t test (*, p < 0.05). The dashed line represents IFN-γ production of cells cultivated in medium (unspecific background).
17, blood was taken from the saphenous vein and serum was
separated from the clotted blood by centrifugation. All animal
experiments were in accordance with local Animal Ethics Com-
mittee regulations.
ELISpot analysis was performed on day 20 after the first
immunization using a mouse IFN-γ ELISpot Kit (R&D Systems,
Minneapolis, MN). Briefly, 2 × 105 spleen cells per well were
stimulated for 24 h in the presence of medium, KLH (10 µg/mL)
or the T cell mitogen concanavalin A (ConA, 10 µg/mL). Spot
development was performed according to the manufacturer’s
instructions and the number of spots was determined using an
ELISpot reader (AID, Straussberg, Germany).
Levels of anti-KLH antibodies in sera of immunized mice were
measured by ELISA. Briefly, Microlon microplates (Greiner,
Frickenhausen, Germany) were coated with 10 µg/mL KLH in 0.05
M Na2CO3 buffer (pH 9.6) at 4 °C overnight. After blocking with
1% BSA/PBS for two hours at rt and washing with 0.05% Tween-
20/PBS plates were incubated with serial dilutions of sera (diluted
in 0.1% BSA/PBS) for two hours. Plates were then washed three
times with 0.05% Tween-20/PBS and incubated with HRP-
conjugated goat-antimouse IgG+A+M antibody in a dilution of
1:1000 (Invitrogen, Basel, Switzerland). Detection was performed
by using the 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate System
(Sigma-Aldrich, Buchs, Switzerland) according to manufacturer’s
instructions.
T Cell Proliferation and ELISpot Analysis. On day 20 after
the first immunization, mice were sacrificed and spleens were
removed. RBCs were lysed by adding hypotonic ammonium
chloride solution. Single cell suspensions were cultivated at 2 ×
105 cells per well in 96-well plates for 24 h in the presence of
medium or KLH (10 µg/mL) for restimulation of T cells ex ViVo.
Proliferation of spleen cells was measured using the CellTiter 96
AQueous One Solution Cell Proliferation Assay (Promega, Madi-
son, WI) according to the manufacturer’s instructions.
Statistical Analysis. Statistical analyses were performed applying
unpaired Student’s t test. All statistical analyses were performed
with the Prism software (Graph Pad Software, San Diego, CA).
Acknowledgment. This research was supported by ETH Zu¨rich,
the Swiss National Science Foundation (SNF Grant 200121-
101593), and the Roche Research Foundation (postdoctoral fel-
lowship for S.B.). S.B. thanks Thailand Research Fund for financial
support.
Supporting Information Available: Complete synthetic pro-
cedures, NMR spectral copies of all new compounds and
complete ref 21. This material is available free of charge via
the Internet at http://pubs.acs.org.
JA806283E
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