Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: Synthesis and biological properties

Article abstract of DOI:10.1016/j.ejmech.2012.02.039

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam₂Cys-α- Galactose as novel immunoadj

Full text of DOI:10.1016/j.ejmech.2012.02.039

European Journal of Medicinal Chemistry 51 (2012) 174e183
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel glycolipid TLR2 ligands of the type Pam2Cys-a-Gal: Synthesis and biological
properties
Jean-Sébastien Thomann a, Fanny Monneaux b, Gaëlle Creusat a, Maria Vittoria Spanedda a,
Béatrice Heurtault a, Chloé Habermacher a, Francis Schuber a, Line Bourel-Bonnet a, Benoît Frisch a
,
*
a Equipe de Biovectorologie, Laboratoire de Conception et Application des Molécules Bioactives, UMR 7199 CNRS/Université de Strasbourg, Faculté de Pharmacie, 74 route du Rhin,
67401 Illkirch Cedex, France
b UPR 9021, Immunologie et Chimie Thérapeutiques, Institut de Biologie Moléculaire et Cellulaire, 15, rue Descartes, 67084 Strasbourg, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation
of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type
Received 21 October 2011
Received in revised form
3 February 2012
Accepted 18 February 2012
Available online 21 March 2012
Pam2Cys-
tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2
agonist activity, by addition of a hydrophilic head, either an -Galactosylpyranose or an -Gal-
a-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic
a
a
actosylfuranose to gain respectively Pam2CGalp and Pam2CGalf. While preparing a carbohydrate building
block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected
galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (a/b ratio > 9) and
Keywords:
Pam2CAG
with good isolated yield (51%). The TLR2 binding properties of Pam2CGalp and Pam2CGalf were then fully
evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared
to that of Pam2CAG and Pam3CAG, two well-established ligands of TLRs. Moreover, the maturation state
of murine dendritic cells previously incubated with either Pam2CGalp or Pam2CGalf was monitored by
flow cytometry and compared to that induced by lipopolysaccharide. Pam2CGalp and Pam2CGalf were
found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very
similar activity, Pam2CGalp and Pam2CGalf were also 10-fold to 100-fold better than Pam2CAG and
Pam3CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to
the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodu-
lating activity. This should greatly enrich the data available on Pam2C structure/activity relationships.
Ó 2012 Elsevier Masson SAS. All rights reserved.
TLR2 agonist
Glycolipid
Immunoadjuvant
DC maturation
1. Introduction
In the field of immunology, the design of adjuvants for vacci-
nation is now shifting ‘from practice to theory’ [1]. Our improved
knowledge of the innate immune system and of the molecular
mechanisms involved in the antigen-specific immune response has
allowed the rational conception of successful vaccine adjuvants. In
addition, in the context of new recombinant protein antigens and to
overcome the poor immunogenicity of certain other antigens like
carbohydrates, potent immunoadjuvants are now deemed essential
with the relevant choice of their structure being the cornerstone of
a successful vaccine strategy.
The stimulation of Toll-Like Receptors (TLRs) present at the
surface of antigen presenting cells (APCs) is now an established
approach to triggering and boosting the immune response. Many
compounds have been described as agonists of the TLRs, especially
the TLR2 subtype [2], and as a consequence, can be considered as
Abbreviations: a-Galf, alpha-D-galactofuranose; a-Galp, alpha-D-galactopyranose;
APC, antigen presenting cell; BOP, benzotriazol-1-yl-oxy-tris-(dimethylamino)-phos-
phonium hexafluorophosphate; CD, cluster of differenciation; DC, dendritic cell; DIEA,
diisopropylethylamine; DMAP, dimethylaminopyridine; DMSO, dimethylsulfoxyde;
EDTA, ethylene diamine tetraacetic acid; ELISA, enzyme linked immunosorbent assay;
FACS, fluorescence-activatedcellsorting; FITC, fluoresceinisothiocyanate;HEK, human
embryonic cells; HRMS, high-resolution mass spectrometry; IFN, interferon; IL, inter-
leukin; LPS, lipopolysaccharide; MALP, macrophage-activating lipopeptide; NF-
kB,
nuclear factor- B; NKT, natural killer T; Pam2CAG, tBoc-[2,3-bispalmitoyloxy-(2R)-
k
propyl]-R-cysteinyl-alanyl-glycine; Pam3CAG, N-Palmitoyl-[2,3-bispalmitoyloxy-(2R)-
propyl]-R-cysteinyl-alanyl-glycine; PBS, phosphate buffer saline; PC, phosphatidyl-
choline; PE, phycoerythrin; PG, phosphatidylglycerol; rmGM-CSF, recombinant mouse
granulocyte/macrophase colony-stimulating factor; SEAPted, secreted embryonic
alkaline phosphatase; SUV, small unilamellar vesicle; TFA, trifluoroacetic acid; THP,
human monocytic leukemia; THP-1, cells; TLR, toll-like receptor; TNF, tumor necrosis
factor.
* Corresponding author. Tel.: þ33 368 854 168; fax: þ33 368 854 306.
E-mail address: frisch@unistra.fr (B. Frisch).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.039

J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
175
potentially interesting immunoadjuvants. For example, very
complex lipopeptides like macrophage-activating lipopeptide-2 [3]
(MALP-2) analogs i.e. Pam2Cys-GDPKHPKSF [4], Pam2Cys-
GDPKHPKSFTGWVA representing the N-terminal part of the 44-
kDa lipoprotein LP44 of Mycoplasma salivarium [5], or Braun lipo-
protein [6] have all been described as TLR2 agonists. On the other
hand, simpler synthetic S-[2,3-bispalmitoyloxy-(2R)-propyl]-R-
cysteinyl lipopeptides, like ‘Pam2CAG’ 1 [7] (Fig. 1) or Pam3CSK4 [8],
have also shown TLR2 agonist activity and can therefore be
considered as more useful and easily attainable immunoadjuvants
[9]. In these constructs, the Pam2C (or Pam3C) template is necessary
but not sufficient to stimulate the TLR2 receptor and a substitution
of the cysteine is required to establish agonist activity. While lysine
derivatives generally improve the activity of TLR2/6 agonists, it was
also shown that Pam2CSK, the smallest lysine-containing
compound, has no cytokine-inducing activity [10]. In this context,
two lysines are usually needed to recover activity, which confer
positive charges and above all highly amphiphilic properties that
complicate the preparation of the vaccine cocktail. Recently,
Pam2CS and its analogs have been synthesized and have shown
blocks based on the presence in glycoconjugates of numerous
microorganisms [14] and absence in mammals of -galactofur-
anosyl residues ( -Galf), presently important targets in anti-
D
D
infectious and immunological chemotherapy research. For
example, beta-galactofuranosyl units are present in microorgan-
isms such as Mycobacterium tuberculosis [15] or Trypanosoma cruzi
[16] in which they play both structural and functional roles and
participate toward our immune defense mechanisms against them
[17]. On the other hand, galactofuranose units in the alpha-
configuration are encountered in Clostridium thermocellum [18],
Penicillium [19], Leishmania [20] and certain pathogenic fungi such
as Paracoccidioides brasiliensis [21] and are also strongly antigenic.
In addition, numerous efforts are devoted to the study of the gal-
actofuranose residue and its derivatives [22]. We therefore
hypothesized that an original double construct, obtained with
a powerful TLR2 agonist tail and a galactosyl head could maintain
and putatively improve its immunoadjuvant properties.
To introduce structural/molecular diversity into the expected
compounds, a convergent strategy was adopted. Thus, the galac-
tosyl head group and the hydrophobic part of the molecule were
each prepared separately only to be coupled during the very last
steps of the synthesis. During the preparation of the sugar building
blocks, we came across a somewhat unexpected high stereo-
selectivity during a halide ion-catalyzed reaction (also known as
the ‘Lemieux reaction’) applied on a protected galactofuranose,
good NF-kB inducing activity [11]. Nevertheless, their effects on DC
and B-lymphocyte maturation and cytokine secretion have not yet
been demonstrated.
In our continuing efforts to rationally design and develop new
glycolipid immunoadjuvants [12], we aimed to synthesize a series
of novel compounds combining a hydrophobic moiety endowed
with TLR agonist properties (TLR2 principally) with a glycosidic
head group found in another family of immunoadjuvants including
yielding predominantly the alpha anomer (
isolated yield (51%). Here we report for the first time the prepara-
tion of the -galactofuranosyl synthon and the stereochemical
a > 90%) with a good
a
a
-galactoceramide [13]. We hypothesized that a glycolipid con-
characterization of this serendipitous result.
structed in such a manner and displaying an original structure
would improve the physico-chemical properties and efficiency of
TLR2 ligands, affording a new family of compounds containing
a hydrophilic head without charge and therefore relatively easy to
The convergent total synthesis of Pam2CGalp and Pam2CGalf
was then performed and the two new highly amphiphilic
compounds, like the references, were either solubilized in a DMSO/
buffer mixture or formulated in liposomes. Their binding to the
synthesize. We first chose to introduce
a
galactose ring as
TLR2 receptor was then studied and compared with that of Pam2
-
a hydrophilic head.
CAG and Pam3CAG. The functional and immunological conse-
quences of the compounds’ agonist activity were investigated.
Finally, Pam2CGalp and Pam2CGalf were found to be 10-fold to 100-
fold better than Pam2CAG and Pam3CAG at inducing B cell prolif-
eration, underlining the importance of this new family of TLR2
agonizing glycolipid immunoadjuvants. Assays to test for CD1d
At this stage of our investigation, the form of the galactose ring,
i.e. pyranose or furanose, was not of relevant importance provided
that both series could be obtained in good yields via unambiguous
synthetic routes. It was decided to use both a-galactosylpyranose
(a
-Galp) and
a
-galactosylfuranose (
a
-Galf) head groups as building
O
O
(CH2)14CH3
(CH2)14CH3
O
O
S
H2N
HO
HO
O
O
C25H51
OH
HO
HN
O
NH
HO
O
O
C14H29
H3C
OH
HN
1
2
COOH
Fig. 1. ‘Pam2CAG’, S-[2,3-bisPalmitoyloxy-(2R)-propyl]-R-cysteinyl-alanyl-glycine 1. ‘
hexacosanoylamino)-1,3,4-octadecanetriol 2.
a-GalCer’ ¼
a
-galactosyl ceramide KRN 7000 ¼ (2S, 3S, 4R)-1-O-(
a-D-galacto-pyranosyl)-2-(N-

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J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
receptor-binding activity displayed by structurally similar gal-
actoceramides such as KRN7000 2 were negative.
In this context, the halide ion-catalytic method developed by
Lemieux [33] is a well-known method to perform 1,2-cis glycosyl-
ations with a large variety of acceptors using galacto- or gluco-
pyranosyl bromides. Lemieux and co-workers investigated this
reaction under neutral conditions and demonstrated that its rate
could be greatly accelerated by addition of tetraethylammonium
bromide. The explanation proposed for this rate increase was based
Here we present in full the successful modification of a TLR2
ligand with a sugar moiety. The structure of the glycolipids is very
simple, non-charged and their adjuvant activity is maintained.
Moreover these results enrich the data available on the structure/
activity relationships of Pam2C-like compounds and support the
development of a new kind of immunomodulating molecule.
on the transient in situ formation of a
more reactive than its counterpart, and which subsequently
undergoes a SN2 displacement by a nucleophilic alcohol to give
selectively the -glycoside. Halide ion catalysis has been shown to
b-glycosyl bromide, which is
a
2. Results and discussion
a
2.1. Chemistry
work using both simple non-hindered alcohols and more complex
selectively protected carbohydrates as acceptors [34].
To achieve the glycolipid compounds, the hydrophilic carbohy-
drate head and the hydrophobic tail were prepared in parallel
before being assembled at the end of the synthesis, according to
a convergent strategy.
We chose to protect
group. The perbenzylation of
D-galactose by a non-participating benzyl
D-galactose 3 (Scheme 1) was per-
formed at 0 ꢀC, by reaction with benzyl bromide after a treatment
by sodium hydride in the presence of catalytic amounts of potas-
sium iodide. Benzyl-2,3,5,6-tetra-O-benzyl-
was exclusively formed, as a viscous oil (67%) [35]. Moreover, only
the
-Galf anomer was isolated, as confirmed by 1H NMR
(J1,2 ¼ 4.2 Hz, compared to a value generally between 3.2 and 3.8 Hz
for C-2 84.2 ppm, compared to a value
-Galp) and 13C NMR (
generally between 76.5 and 77.5 ppm for -Galp). Bordoni et al. [36]
found similar results when synthesizing galactofuranosidase
substrates. In addition, while preparing perbenzylated galactose by
using an excess of potassium hydroxide in DMSO, Decoster et al.
[37] also demonstrated by NMR that the sugar was obtained in
a-D-galactofuranoside 4
2.1.1. Preparation of the
2.1.1.1. -Galactofuranose head: an unexpected highly alpha-selective
halide ion-catalyzed reaction. To achieve the sugar head group
synthesis, we first investigated the chemistry of -galactose and
especially 1,2-cis O-glycosidation. In the literature, the use of galac-
tosyl pyranose donors in 1,2-cis O-glycosidation reactions is well-
established and numerous methods have been reported using tri-
chloroacetimidates [23], thioglycosides [24] or halide glycosides [25]
protected in the 2-position with non-participating protecting groups
such as glycoside donors. In contrast, fewer methods are available to
D-galactosyl heads
a
a
D
a
d
a
excellent yield as the a-furanose form.
obtain 1,2-cis linkages for
because furanosides present generally a lower anomeric effect [26],
rendering the preparation of -Galf derivatives more difficult than
that of their pyranosyl ( -Galp) counterparts. -Galf activated as tri-
chloroacetimidates have been used in several studies [27], however
the occurrence of many side products and only limited stereo-
selectivities were reported. Encouraging results have, however, been
described starting from 2-O-benzylated 1,2-trans-thiogalactofurano-
sides, however the stereoselectivity of the coupling reaction appeared
to be strongly dependent on the nature of the nucleophilic alcohol
D
-galactose in its furanose form, probably
Next, a careful treatment by acetic acid at 80 ꢀC in the presence
of either sulfuric acid or triflic acid afforded the sole deprotection of
the anomeric hydroxyl (C-1). The conditions of the reaction were
finely tuned to avoid further deprotection of the other alcohols,
a side reaction already described for pyranosyl glycosides in the
D
D
D
literature [38]. The product 5 was isolated as a mixture of a- and b-
anomers (1:1). Treatment of 5 by acetic anhydride in the presence
of 1 equiv of pyridine and catalytic amounts of N,N-dimethyl-4-
aminopyridine (DMAP) was performed under argon. The selec-
tively mono-acetylated compound 6 was obtained with 96% yield
[28]. Later, the synthesis of 2,3,5,6-tetra-O-benzyl-
was described to obtain allyl- -galactofuranoside as a precursor of
galactofuranose-containing oligosaccharides [29]. After that, the
synthesis of -galactofuranosyl-based oligosaccharides was
D
-galactofuranose
and a
-compound had a very small J1,2 coupling constant whereas that of
the corresponding alpha anomer ( 6.25 ppm) was 4.2 Hz.
a/b ratio of 8.5/1.5. The anomeric proton (d 6.28 ppm) for the
a-D
b
d
a-D
Finally, a halide ion-catalyzed reaction was undertaken to obtain
permitted by the selective glycosylation of an aldono-1,4-lactone by
the trichloroacetimidate method [30]. Another interesting strategy to
compound 8 (Scheme 1). To this end, a solution of 33% hydrogen
bromide in acetic acid was added to 6. The bromo sugar in its b form
was too unstable to be isolated and its fast deterioration on silica gel
hampered the monitoring of the reaction by TLC. The major isomer
obtained during this reaction could nevertheless be identified by 1H
achieve
a
-galactofuranosylation is the use of 20-carboxybenzyl
glycosides as glycosyl donors [31] and some examples of synthesis
involving
a-D-galactofuranosides are now described [32].
HO
HO
OBn
OBn
O
O
O
b)
a)
OH
OH
HO
OBn
OBn
HO
OBn
OBn
OBn
OBn
OBn
3
4
5
OBn
OBn
O
O
c)
d), e)
OAc
O
O
OBn
OBn
OBn
OBn
OBn
OBn
NHBoc
H
NHBoc
6
7
8
Scheme 1. Synthesis of galactofuranosyl head 8. a) BnBr excess, NaH, KI, DMF (67%); b) AcOH, 1.2 M H2SO4 (81%) or 1 M Triflic acid, AcOH (60%)
a/b 1:1; c) Ac2O, pyridine, DMAP
(96%) ratio 8.5:1.5; d) HBr in AcOH, CH2Cl2, rt, 10 min; e) tetrabutylammonium bromide, 4 Å molecular sieves, nucleophilic alcohol (7), DIEA, CH2Cl2, rt, 2 days.
a/b

J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
177
NMR. The
over 90% of the reaction products, whereas only traces of
furanose were observed (d,
6.65 ppm, J1,2 ¼ 4.2 Hz) (see Supporting
b
-bromofuranose anomer (s,
d
6.50 ppm) accounted for
overall yield starting from the
a
-galactopyranosyl intermediate 12.
a-bromo-
For Pam2CGalp 18, comparable overall yields (28%) were obtained.
These are normal yields for complex amphiphilic compounds the
synthesis and purification of which are renowned as being difficult.
During this synthetic part, we observed a serendipitous selec-
tivity during a halide ion-catalytic reaction to obtain a
a galactofuranosyl derivative. This short synthesis (4 steps from
galactose) gives a very convenient access to a -Galf building block
in good yield (51%) and with a high stereoselectivity ( ratio > 9).
From a mechanistic viewpoint, the stereochemical outcome of this
d
information). Other minor side products were found such as hydro-
lysis products or starting materials. No 13C NMR analysis could be
performed on this bromo sugar due to its poor stability over the time
needed for analysis. After 10 min of reaction, reagents and solvent
were removed by co-evaporation with anhydrous toluene. The
mixture was then dissolved in CH2Cl2, followed by the addition of
molecular sieves (4 Å) and tetrabutylammonium bromide. After
5 min, under stirring, tert-butyl 2-hydroxyethylcarbamate 7 was
added in the presence of diethylisopropylamine and the reactionwas
left for 2 days at r.t. After purification, the major isomer was fully
a-linkage in
D
-
a
a/b
reaction could be due to the formation of a
followed by a direct SN2 reaction.
b-bromoGalf derivative
assigned as the
a-galactofuranosyl N-Boc protected amine 8 (51%)
2.2. Biology
with a high anomeric ratio (a/b
> 9). This relatively quick synthesis
led to the compound 8 with an overall isolated yield of 28%. In
addition, this approach is of particular interest since it affords almost
exclusively the alpha anomer.
Glycolipids 17 and 18 e named Pam2CGalf and Pam2CGalp,
respectively e were then studied with regards to their biological
properties in comparison with two well-established ligands of
TLR2, Pam2CAG and Pam3CAG. The usual conditions used for the
easy handling of such amphiphilic compounds including their
solubilization in DMSO/buffer mixture or incorporation into lipo-
somes were applied in this work and are specified hereafter.
2.1.1.2.
a-Galactopyranose head. The corresponding pyranose
analog was also synthesized (Scheme 2). Starting compound 9 was
obtained according to the procedures described by Nicolaou et al.
[39]. Next, the anomeric thiophenyl group was hydrolyzed in the
presence of N-bromosuccinimide to get 10. A final acetylation and
a halide ion-catalytic reaction were performed (under the same
conditions as those reported above for compound 6, Scheme 1) to
selectively give compound 12 with a 48% overall yield in 3 steps. A
2.2.1. Demonstration of the TLR2 binding and agonist activity
We tested compounds Pam2CGalp and Pam2CGalf as liposome
suspensions for TLR agonist activity using the cell line THP1-BlueÔ
CD14, that reports TLR1 to TLR10 activities (with the exception of
small proportion of
b-isomer (8%) could be easily separated by
TLR3 and TLR9) via NF-kB transcription of secreted embryonic
chromatography on silica gel.
alkaline phosphatase (SEAP) (Fig. 2A). To determine the selectivity
of our compounds, we also performed similar experiments while
neutralizing the TLR2 receptor with a selective monoclonal anti-
body directed against the TLR2 receptor (Fig. 2B).
2.1.2. Assembling the lipopeptidic core
The Boc-protecting group of compounds 8 and 12 was removed
using trifluoroacetic acid in dichloromethane in quantitative yields.
The corresponding primary amines 13 and 14 were coupled to N-
Boc-S-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteine (the synthesis
of which has already been described) [7] using Castro’s reagent
The agonist activity of Pam2CGalp and Pam2CGalf was thus
found to be as high as that of Pam2CAG (0.1
mM) and 100-fold
higher than Pam3CAG (10 M). Regarding the preparation, the
m
same activity was observed when the compounds were either
formulated in liposomes or dissolved in PBS containing 2% of DMSO
(data not shown). When the TLR2 receptor was neutralized (open
bars), no significant agonist activity was observed with the four
compounds, thus demonstrating their TLR2 receptor selectivity.
This TLR2 agonist activity was confirmed using another cell line
expressing only the TLR2 receptor (HEK-Blue-2 Cells; InvivoGen,
Toulouse, France) (data not shown).
(benzotriazol-1-yl-oxy-tris-dimethylaminophosphonium
tetra-
fluorophosphate, BOP) in the presence of DIEA in dichloromethane
(Scheme 3). Intermediate amides 15 and 16 were obtained with
good yields, 69% and 62% respectively. The deprotection of the
alcohol functions that followed consisted in the removal of every
benzyl group by hydrogen in the presence of carbon-supported
palladium catalyst. The catalytic hydrogenation delivered the
desired products in relatively low yields (40% and 47% respectively),
probably due to the poisoning of the catalyst by the cysteinyl sulfur
atom, or to the amphiphilic nature of the compound. Finally, the
tBoc protection was removed using 5% TFA in dichloromethane to
obtain the expected compounds with high yields (70% and 96%
respectively). Thus, Pam2CGalf 17 was attained in 3 steps with 20%
2.2.2. Demonstration of the functional activity of Pam2CGalp and
Pam2CGalf
2.2.2.1. Maturation of DCs. Using the mouse DC line D1, we then
investigated the capacity of TLR agonists to induce DC maturation.
As shown in Fig. 3, the two new TLR agonists up-regulated the
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
b)
a)
O
O
O
SPhe
OH
OAc
BnO
BnO
BnO
BnO
9
10
11
BnO
BnO
O
c), d)
BnO
NHBoc
O
BnO
H
O
7
12
NHBoc
Scheme 2. Synthesis of galactopyranosyl head 12. a) N-bromosuccinimide, acetone, H2O (99%); b) Ac2O, pyridine, DMAP (96%); c) HBr in AcOH, CH2Cl2, rt, 10 min; d) tetrabuty-
lammonium bromide, 4 Å molecular sieves, nucleophilic alcohol (7), DIEA, CH2Cl2, rt, 2 days.

178
J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
OBn
O
BnO
BnO
O
O
OBn
OBn
OBn
BnO
NH2
BnO
O
14
NH2
13
a)
a)
BnO
BnO
Pam
O
Pam
O
O
O
O
OBn
O
BnO
O
Pam
O
Pam
BnO
O
O
OBn
OBn
S
NHBoc
N
H
OBn
N
H
S
NHBoc
16
15
b), c)
b), c)
HO
Pam
O
Pam
HO
HO
O
O
O
OH
O
O
O
Pam
O
Pam
HO
O
O
OH
OH
S
OH
N
H
S
N
H
NH2
NH2
Pam2CGalp, 18
Pam2CGalf, 17
Scheme 3. Synthesis of Pam2CGalf, 17 and Pam2CGalp, 18. a) N-a-tBoc-S-[2,3-bis(palmitoyloxy)-2R)-propyl]-L-cysteine, BOP, DIEA, CH2Cl2; b) H2, Pd/C, MeOH, CH2Cl2; c) TFA/CH2Cl2
50/50 (v/v).
expression of costimulatory molecules (maturation markers), such
Galceramides (known to bind CD1d molecules) and thus activate
NKT cells. While all four compounds induced the proliferation of
splenocytes from BALB/c mice, no cytokines typically produced by
as CD40, CD54, CD80 and CD86, to levels comparable with Pam2
-
CAG and Pam3CAG. It is noteworthy that each compound (used at
2.5
mg/mL, as DMSO/PBS 2/98 solution) induced a maturation level
activated NKT cells (IL-4 and IFN-g) were detected. Moreover, the
similar to that induced by LPS. Moreover, such maturation was
observed in the presence of polymyxin B, an inhibitor of LPS
activity, thus ruling out the possibility of a LPS-mediated matura-
tion of D1 cells (Fig. 3).
proliferative response was not inhibited by a blocking mAb directed
against the CD1d molecule (data not shown). These results show
that the mechanism used by the two new immunoadjuvants is
independent of CD1d.
2.2.2.2. Proliferation of immune cells. Finally, we evaluated the
functional properties of Pam2CGalp and Pam2CGalf by examining
their capacity to activate splenocytes from naive mice ex vivo. BALB/c
splenocytes were incubated ex vivo with increasing doses of Pam2C-
Galp, Pam2CGalf, Pam2CAG or Pam3CAG ranging from 0 to 1000 ng/
mL, and evaluated for their capacity to proliferate and secrete cyto-
kines. Cell proliferation was measured after 3 days by tritiated
thymidine incorporation and cytokine production was determined in
culture supernatants. Results showed that the four agonists induced
cellproliferation (Fig. 4A)thatwasaccompanied bya dose-dependent
3. Conclusion
Here we have characterized for the first time two TLR2 ligands
that have been modified by a sugar moiety. In addition, we have
revealed that the Lemieux reaction can lead to obtaining a aGalf
synthon with good yields and a serendipitous stereoselectivity
toward the alpha anomer. These new synthetic amphiphilic
immunoadjuvants display potent immune activity. Both bind TLR2
and are capable of triggering dendritic cell maturation and B cell
activation. Not only does this work greatly enrich the data available
on Pam2C structure/activity relationships, but it could also open the
way for an interesting new class of immunoadjuvants.
secretion of IL-6 and TNF-a by BALB/c splenocytes (Fig. 4B).
As TLR2 is largely expressed on B cells, we repeated this
experiment by using purified B cells. As expected, all four TLR
agonists strongly activated B cells leading to their dose-dependent
proliferation (Fig. 5). Noteworthy however was the apparent
increased potency of the new compounds Pam2CGalp and Pam2C-
Galf compared to Pam3CAG and Pam2CAG as shown by a higher
4. Experimental protocols
4.1. Chemistry
proliferative response at low doses (0.001
Since our new analogs possess a -galactopyranose or
actofuranose ring, we evaluated whether they could mimic
m
g/mL; Fig. 5).
4.1.1. General chemical procedures
All starting materials were obtained from commercial suppliers
and were used without further purification. All reactions were
a
a
-gal-
a
-

J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
179
Fig. 2. Quantification of TLR agonist activity by SEAP activity of THP1-Blue-CD14 cells. A: THP1-Blue-CD14 cells were incubated for 19 h with increasing amounts of Pam2CAG,
Pam3CAG, Pam2CGalp and Pam2CGalf formulated in PC/PG/Chol liposomes. The resulting SEAP activity was compared to that of cells incubated with lipopolysaccharide (LPS) (1 g/
well) (positive control, plain horizontal line), or blank liposomes (negative control, doted horizontal line). B: The TLR2 receptor of THP1-BlueÔ CD14 cells was neutralized using
100 ng of an anti-mTLR2 antibody 10 min before addition of 0.1 M of putative agonist in liposomal samples (except for Pam3CAG used at 10 M because no significant SEAP activity
m
m
m
was detected under this threshold). The SEAP activity (open bars) was compared to that of cells without antibody (plain bars). Error bars represent the standard deviation of
experiments performed in triplicate.
Fig. 3. Maturation of the mouse dendritic cell line D1. Expression of maturation markers (CD40, CD54, CD80 and CD86), as measured by flow cytometry after 48 h of incubation with
Pam2CGalp (green), Pam2CGalf (pink), Pam3CAG (purple) or Pam2CAG (yellow) (2.5
mg/mL). LPS (10 mg/mL) was used as control without Polymyxin B (P) (blue). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)

180
J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
Fig. 4. Activation in response to ex vivo stimulation with the four TLR2 agonists. Splenocytes (2 ꢂ 105/well) from BALB/c mice were cultured with increasing doses of agonist. (A) Cell
proliferation was assessed by [3H]-thymidine incorporation at day 3 and the results are expressed as stimulation index (SI) ꢃ SD of triplicate wells, corresponding to the ratio of
counts per minute (cpm) in the medium with the studied agonist to cpm in the medium without the agonist. (B) IL-6 and TNF-a levels were measured by double-sandwich ELISA in
30 h-supernatants and results are expressed as the mean concentration (pg/mL) ꢃ SD of duplicates.
carried out under argon. Dry solvents were used in all experiments.
Thin-layer chromatography was performed on Merck-precoated
RP-18 F254S plates and compounds were observed by UV or by
CH2Cl2 (50 mL). After 24 h stirring, the organic phase was washed
with brine (20 mL), dried with MgSO4, and after filtration and
evaporation to dryness, the residue was purified by column chro-
matography on silica gel (cyclohexane/EtOAc: 7.5/2.5) to give
compound 15 (1.3 g, 69%). Rf 0.5 (cyclohexane/EtOAc: 8/2). 1H NMR
spraying a solution of
a
-naphtol. Flash column chromatography
m) were carried out with
separations (silica gel 60 Merck, 40e60
m
cyclohexane-ethyl acetate mixtures as eluent. 1H NMR and 13C NMR
spectra were recorded on Bruker Advance DPX300 (300 MHz) and
DPX400 (400 MHz) spectrometers. The NMR chemical shifts are
reported in ppm relative to chloroform using its residual proton for
1H NMR spectra and its carbon atom for 13C NMR spectra. High-
resolution mass spectrometry was performed on an Agilent MSD
with Agilent 1200 SL HPLC equipped with DAD. Optical rotations
were measured on a P-2000 Jasco polarimeter at 20 ꢀC and are
(300 MHz, CDCl3): d 7.44e7.17 (m, 20H, Ph), 6.90 (t, 1H, J 6.2 Hz,
NHCO), 5.37 (bs, 1H, NHBoc), 5.16e5.09 (m, 1H, CHO), 4.78 (d, 1H, J
4.2 Hz, CH2Pam), 4.73 (d, 1H, J 11.4 Hz, CH2-Ph), 4.70 (d, 1H, J
11.4 Hz, CH2-Ph), 4.65 (d, 1H, J 12.3 Hz, CH2-Ph), 4.59 (d, 1H, J
12.3 Hz, CH2-Ph), 4.51 (s, 2H, CH2-Ph), 4.47 (d, 1H, J 11.4 Hz, CH2-
Ph), 4.43 (d, 1H, J 11.4 Hz, CH2-Ph), 4.32 (t, 1H, J 7.4 Hz, H-3), 4.29
(dd, 1H, J 4.2 Hz, J 12.4 Hz, CH2Pam), 4.16e4.13 (m, 1H, CHCO), 4.12
(dd, 1H, J 6.4 Hz, J 12.4 Hz, CH2O glycerol), 4.00 (dd, 1H, J 4.6 Hz, J
7.9 Hz, H-2), 3.96 (dd, 1H, J 7.3 Hz, J 1.9 Hz, H-4), 3. 71e3.64 (m, 3H,
H-6, H-5), 3.56 (t, 2H, J 5.0 Hz, CH2O), 3.36 (dd, 2H, J 5.0 Hz, J 5.0 Hz,
CH2N), 2.80e2.70 (m, 2H, CH2SCys), 2.67 (d, 2H, J 6.5 Hz, CH2S), 2.30
(t, 4H, J 7.5 Hz, CH2Pam), 1.64e1.59 (m, 4H, CH2Pam), 1.43 (s, 9H,
tBu), 1.26 (bs, 48 H, CH2Pam), 0.88 (t, 6H, J 6.8 Hz, CH3Pam); 13C
given in 10ꢁ1 deg cm2 gꢁ1
.
4.1.2. Synthesis of Pam2CGalf (17)
4.1.2.1. Synthesis of [2,3-bis(palmitoyloxy)-(2R)-propyl]-N-(tert-
butoxycarbonyl)-(R)-cysteinyl-amino-ethyl-O-(2,3,5,6-tetra-O-benzyl-
a-D
-galactofuranose) 15. A solution of 8 (1 g, 1.46 mmol) in CH2Cl2/
NMR (75 MHz, CDCl3): d 173.9 (COO), 173.6 (COO), 170.8 (NHCO),
TFA 8/2 (10 mL) was stirred for 1 h before being evaporated to give
compound 13 (1 g, 98%) as a TFA salt. This was then added without
prior purification (0.82 mg, 1.4 mmol) with DIEA (0.84 mL,
5.1 mmol) to a solution of N-Boc-S-[2,3-bispalmitoyloxy-(2R)-
propyl]-R-cysteine (1.1 g, 1.4 mmol) and BOP (0.75 g, 1.7 mmol) in
156.0 (NHCOOtBu),138.7e138.2 (Cq Ph),129.2e128.3 (CH Ph),100.1
(C-1), 84.5 (C-2), 80.5 (C-3, Cq tBu), 80.3 (C-4), 78.1 (C-5), 73.4e72.3
(CH2-Ph), 70.8 (CHO), 70.5 (C-6), 68.6 (CH2O), 64.2 (CH2OGlycerol),
55.0 (CHNH), 40.1 (CH2NH), 36.2 (CH2SCys), 34.9 (CH2Pam), 34.7,
33.7 (SCH2), 32.5 (CH2Pam), 30.3e29.8 (CH2Pam), 28.9 (tBu), 25.5
(CH2Pam), 23.3 (CH2Pam), 14.75 (CH3Pam); HRMS (ESI) calcd for
C79H120N2NaO13S [M þ Na]: 1359.8427; found: 1359.8403.
4.1.2.2. Synthesis of [2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cys-
teinyl-amino-ethyl-O-(a-D-galactofuranose) (Pam2CGalf, 17). Pd/C
(112 mg) was carefully added to a solution of compound 15
(580 mg, 0.433 mmol) in CH2Cl2/MeOH 1/1 (15 mL). The reaction
medium was stirred under hydrogen atmosphere at 60 psi. After 3
days, the Pd/C was filtered on celite and rinsed several times with
THF, then with a mixture of CH2Cl2/MeOH: 5/5. The organic phase
was evaporated under vacuum and the crude product was purified
on silica gel (CH2Cl2/MeOH: 9/1) to give the unbenzylated product
(170 mg, 40%). To remove the Boc-protecting group, a solution of
unbenzylated compound (100 mg, 0.102 mmol) in CH2Cl2/TFA: 5/1
(10 mL) was stirred under argon for 1 h then evaporated under
vacuum to give Pam2CGalf as the corresponding trifluoroacetate
salt. The crude was purified on silica gel (CH2Cl2/MeOH: 9/1 to 8.5/
Fig. 5. B cell proliferation in response to ex vivo stimulation with the four agonists.
Purified B cells (1 ꢂ105/well) from BALB/c mice were cultured with increasing doses of
the four agonists. Cell proliferation was assessed by [3H]-thymidine incorporation at
day 3 and the results are expressed as stimulation index (SI) ꢃ SD of triplicate wells,
corresponding to the ratio of counts per minute (cpm) in the medium with the studied
agonist to cpm in the medium without the agonist.
1.5) to give the compound Pam2CGalf as a white solid (71 mg, 70%;
20
overall yield 20%). [
a
]
D ꢁ6.9 (c 0.11, CHCl3). Rf 0.15 (CH2Cl2/MeOH:
5.16e5.10 (m, 1H,
CHO), 4.81 (d, 1H, J 4.7 Hz, H-1), 4.30 (dd, 1H, J 11.8 Hz, J 3 Hz,
9/1). 1H NMR (300 MHz, CDCl3/MeOD 9/1):
d

J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
181
CH2OGlycerol), 4.10 (t, 1H, J 7.3 Hz, CHNH2), 4.05 (dd, 1H, J 6.4 Hz, J
11.8 Hz, CH2OGlycerol), 3.93 (dd, 1H, J 4.7 Hz, J 8 Hz, H-2), 3.75 (dd,
1H, J 5.5 Hz, J 10.6 Hz, H-6), 3.69 (dd, 1H, J 6.6 Hz, J 10.6 Hz, H-6),
3.62 (m, 2H, H-4, H-3), 3.54 (t, 2H, J 5.3 Hz, CH2O), 3.49 (m, 1H, H-5),
3.39 (t, 2H, J 5.3 Hz, CH2N), 2.93 (dd, 1H, J 5 Hz, J 13.5 Hz, CH2SCys),
2.80e2.60 (m, 3H, CH2SCys, CH2S), 2.25 (dd, 4H, J 4.7 Hz, J 7.5 Hz,
CH2Pam), 1.64e1.60 (m, 4H, CH2Pam), 1.18 (bs, 48H, CH2Pam), 0.78
(t, 6H, J 6.8 Hz, CH3Pam); 13C NMR (75 MHz, CDCl3/MeOD 9/1):
CH2Cl2/TFA: 5/1 (10 mL) was stirred under argon for 1 h before
being evaporated under vacuum to give Pam2CGalp as the corre-
sponding trifluoroacetate salt. The crude product was purified on
silica gel (CH2Cl2/MeOH: 9/1 to 8.5/1.5) to give the compound
Pam2CGalp as a white solid (120 mg, 96%, 28% overall yield).
20
[a
]
þ28.5 (c 0.11, CHCl3). Rf 0.15 (CH2Cl2/MeOH: 9/1). 1H NMR
D
(400 MHz, CDCl3/MeOD 9/1):
d 5.08 (m, 1H, CHO), 4.76 (d, 1H, J
2.8 Hz, H-1), 4.27 (dd, 1 H, J 12 Hz, J 3 Hz, CH2OGlycerol), 4.02 (dd,
1H, J 6.4 Hz, J 12 Hz, CH2OGlycerol), 4.03 (m, 1H, CHN), 3.83 (s, 1H,
H-4), 3.80e3.60 (m, 6H, CH2O, H-3, H-2, H-6, H-5), 3.47 (dd, 1H, J
7.7 Hz, J 2 Hz, CH2N), 3.43 (dd, 1H, J 7.7 Hz, J 4 Hz, CH2N), 3.30 (m,
1H, H-6), 2.98 (dd,1H, J 6 Hz, J 14 Hz, CH2SCys), 2.84 (dd,1H, J 7 Hz, J
14 Hz, CH2SCys), 2.70 (dd, 1H, J 6 Hz, J 14 Hz, CH2S), 2.63 (dd, 1H, J
7.7 Hz, J 14 Hz, CH2S), 2.22 (q, 4H, J 7 Hz, CH2Pam), 1.50 (m, 4H,
d
174.2 (COO), 173.7 (NCO), 173.4 (COO), 102.1 (C-1), 72.3, 75.2, 77.9,
82.9 (C-5, C-4, C-3, C-2), 70.8 (CHO), 68.2 (CH2O), 64.4 (C-6), 64.2
(CH2OGlycerol), 54.6 (CHNH2), 39.9 (CH2NH), 38.2 (CH2SCys),
34.8e34.6 (CH2Pam), 33.0 (SCH2), 32.4 (CH2Pam), 30.0e29.6
(CH2Pam), 25.4 (CH2Pam), 23.2 (CH2Pam), 14.6 (CH3Pam); HRMS
(ESI) calcd for C46H89N2O11S [M þ H]: 877.6182; found: 877.6198.
CH2Pam), 1.16 (m, 48H, CH2Pam), 0.78 (t, 6H, J 6.8 Hz, CH3Pam); 13
NMR (100 MHz, CDCl3/MeOD 9/1): 174.3 (COO),173.4 (COO), 168.2
C
4.1.3. Synthesis of Pam2CGalp (18)
d
4.1.3.1. Synthesis of [2,3-bis(palmitoyloxy)-(2R)-propyl]-N-(tert-
butoxycarbonyl)-(R)-cysteinyl-amino-ethyl-O-(2,3,5,6-tetra-O-benzyl-
a-D-galactopyranose) (16). A solution of 12 (1 g, 1.46 mmol) in
(NCO), 99.5 (C-1), 71.4e70.1 (C-5, C-4, C-3, C-2), 69.4 (CHO), 67.1
(CH2O), 64.3 (CH2OGlycerol), 62.3 (C-6), 52.7 (CHNH2), 40.2
(CH2NH), 34.7e34.5 (CH2Pam), 33.3 (CH2SCys), 32.6 (CH2S), 32.3
(CH2Pam), 30.1e29.5 (CH2Pam), 25.4e25.3 (CH2Pam), 23.1
CH2Cl2/TFA 8/2 (10 mL) was stirred for 1 h before being evaporated
to give compound 14 (1 g, 98%) as a TFA salt. This was then added
without purification (323 mg, 0.55 mmol) with DIEA (0.345 mL,
2.1 mmol) to a solution of N-Boc-S-[2,3-bispalmitoyloxy-(2R)-
propyl]-R-cysteine (420 mg, 0.55 mmol) and BOP (310 mg,
0.7 mmol) in CH2Cl2 (25 mL). After 24 h stirring, the organic phase
was washed with brine (20 mL), dried with MgSO4, and after
filtration and evaporation to dryness, the residue was purified
by column chromatography on silica gel (cyclohexane/EtOAc:
7.5/2.5) to give compound 16 (420 mg, 62%). Rf 0.5 (cyclohexane/
(CH2Pam), 14.4 (CH3Pam); HRMS (ESI) calcd for C46H89N2O11
S
[M þ H]: 877.6182; found: 877.6197.
4.2. Biology
4.2.1. Liposome preparation
Liposomes were prepared by mixing phospholipids (PC, PG),
cholesterol and lipopeptides or glycolipids (78/20/50/2 molar ratio)
dissolved in chloroform/methanol (9:1, v/v) in a round-bottom
flask. After solvent evaporation under high vacuum, the dried
lipid film was hydrated by vortex mixing after addition of 1 mL of
sterile PBS (Eurobio, Courtaboeuf, France). The final concentration
EtOAc: 8/2). 1H NMR (300 MHz, CDCl3/MeOD 9/1):
d 7.45e7.20 (m,
20 H, Ph), 6.99 (t, 1H, J 5.7 Hz, NHCO), 5.37 (bs, 1H, NHBoc),
5.16e5.10 (m, 1H, CHO), 4.92 (d, 1H, J 11.8 Hz, CH2-Ph), 4.86 (d, 1H, J
11.8 Hz, CH2-Ph), 4.84 (d, 1H, J 11.8 Hz, CH2-Ph), 4.78 (d, 1H, J 3.7 Hz,
H-1), 4.74 (d, 1H, J 11.8 Hz, CH2-Ph), 4.65 (d, 1H, J 11.8 Hz, CH2-Ph),
4.55 (d,1H, J 11.8 Hz, CH2-Ph), 4.47 (d,1H, J 11.8 Hz, CH2-Ph), 4.42 (d,
1H, J 11.8 Hz, CH2-Ph), 4.27 (dd, 1H, J 3.5 Hz, J 11.8 Hz, CH2OGly-
cerol), 4.16 (m, 1H, CHNH), 4.09 (dd, 1H, J 6.2 Hz, J 11.8 Hz, CH2O-
Glycerol), 4.03 (dd,1H, J 9.3 Hz, J 3.7 Hz, H-2), 4.00 (t,1H, J 6.5 Hz, H-
5), 3.93 (dd, 1H, J 2.7 Hz, J 9.3 Hz, H-3), 3.91 (apparent s, 1H, H-4),
3.80 (m, 1 H, CH2O), 3.72 (m, 1H, CH2O), 3.64 (dd, 1H, J 9.8 Hz, J
6.3 Hz, H-6), 3.56 (dd, 1H, J 9.8 Hz, J 6.8 Hz, H-6), 3.44 (m, 2H,
CH2NH), 2.80e2.70 (m, 2H, CH2SCys), 2.68 (d, 2H, J 6.5 Hz, CH2S),
2.28 (t, 4H, J 7.5 Hz, CH2Pam), 1.64e1.59 (m, 4H, CH2Pam), 1.40 (s,
of phospholipids was 10 mmol/mL. The resulting suspension was
sonicated at 25 ꢀC for 50 min using a 3 mm diameter probe soni-
cator (Vibra Cell, Sonics and Material Inc., Danbury, CT) at 300 W
(5 s cycles interrupted for 1.25 s). The resulting Small Unilamellar
Vesicles (SUV) were finally centrifuged for 10 min at 10,000 g to
remove the titanium dust originating from the probe. The average
diameter of the liposomes was determined by dynamic light scat-
tering using a NanoZS apparatus (Malvern Instrument, Orsay,
France). The different vesicle preparations were homogeneous in
size and exhibited an average diameter between 80 and 110 nm.
9H, tBu), 1.25 (bs, 48H, CH2Pam), 0.88 (t, 6H, J 6.8 Hz, CH3Pam); 13
NMR (75 MHz, CDCl3/MeOD 9/1): 173.9 (COO), 173.7 (COO), 170.9
C
4.2.2. In vitro determination of TLR stimulation
d
We used the human peripheral blood monocytic cell line THP1-
BlueÔ-CD14 cells (InvivoGen, Toulouse, France) that express TLRs 1,
2, 4e8 and 10 and are transfected with a reporter plasmid con-
taining secreted embryonic alkaline phosphatase (SEAP) under the
control of an NF-kB promoter. These THP1-BlueÔ-CD14 cells were
cultured in complete RPMI 1640 medium containing glucose
(NHCO), 155.9 (NHCOOtBu), 139.3e 138.1 (4Cq Ph), 129.1e128.1 (CH
Ph), 99.2 (C-1), 84.5 (C-2), 79.8 (Cq tBu), 77.9 (C-3), 77.1 (C-2), 75.7
(C-4), 75.3e74.0 (CH2-Ph), 70.8e70.7 (C-5, CH2O, CHO), 69.2 (C-6),
64.2 (CH2OGlycerol), 54.6 (CHNH), 40.3 (CH2NH), 36.1 (CH2SCys),
34.9e34.7 (CH2Pam), 33.6 (CH2S), 32.6 (CH2Pam), 30.4e29.8
(CH2Pam), 29.0 (tBu), 25.6e25.5 (CH2Pam), 23.4 (CH2Pam), 14.8
(4.5 mg/mL) and supplemented with 10% endotoxin-free FBS
(CH3Pam); HRMS (ESI) calcd for C79H120N2NaO13
1359.8427; found: 1359.8403.
S
[M þ Na]:
(Lonza, Levallois-Perret, France), 100 units/mL penicillin, 100
streptomycin (Eurobio, Courtaboeuf, France), 200 g/mL zeocin and
10 g/mL blasticidine (both from InvivoGen, Toulouse, France).
mg/mL
m
m
4.1.3.2. Synthesis of [2,3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cys-
teinyl-amino-ethyl-O-( -galactopyranose) (Pam2CGalp, 18). Pd/C
Cells were maintained at 37 ꢀC in a 5% CO2 humidified atmosphere.
TLR agonist activity of our different compounds was determined
on THP1-BlueÔ-CD14 cells by quantifying SEAP activity using the
Quanti-BlueÔ method (InvivoGen, Toulouse, France). Briefly, cells
a-D
(100 mg) was carefully added to a solution of compound 16
(400 mg, 0.3 mmol) in CH2Cl2/MeOH: 1/1 (15 mL). The reaction
medium was stirred under hydrogen atmosphere at 60 psi. After 3
days, the Pd/C was filtrated on celite and rinsed several times with
THF, then with a mixture of CH2Cl2/MeOH: 5/5 v/v. The organic
phase was evaporated under vacuum and the crude product was
purified on silica gel (CH2Cl2/MeOH: 9/1) to give the unbenzylated
product (140 mg, 47%). To remove the Boc-protecting group,
a solution of unbenzylated compound (140 mg, 0.143 mmol) in
were plated at 1.5 ꢂ106 cells/mL in a 96-well plate (100
mL of
complete medium), and incubated with 10 mL of the different
liposomal samples at 37 ꢀC. Cells were stimulated with the TLR4
agonist LPS (1 g) as a positive control and with PC/PG/cholesterol
(80/20/50) liposomes as a negative control. After 19 h of incubation,
20 L of supernatant fluid was removed and added to 200 L of the
Quanti-BlueÔ colorimetric assay reagent in each well of a 96-well
m
m
m

182
J.-S. Thomann et al. / European Journal of Medicinal Chemistry 51 (2012) 174e183
plate for 2 h at 37 ꢀC. The optic density was then read at 595 nm
using a microplate reader (Bio-Rad model 550, Marnes-la-Coquette,
France). To determine the selectivity of our compounds, we per-
formed the same experiments incubating cells with 100 ng/well of
a monoclonal anti-mTLR2 antibody (InvivoGen, Toulouse, France)
for 10 min prior to the addition of samples.
2 ꢂ 105 cells/mL were cultured for 48 h. Then, fresh medium con-
tainingtheTLRanalogs wasadded. All experiments(except whenLPS
was added) were performed in the presence of polymyxin B at 20 mg/
mL. After 48 h of incubation, cells were washed with cold PBS and
then harvested with 2 mL of PBS containing 2 mM EDTA. After
centrifugation, cells were suspended again in PBS containing 2% FCS
and then incubated at 4 ꢀC for 20 min with the various antibodies
used at a concentration recommended by the manufacturer. After
two washes in PBSe2% FCS, cells were acquired and analyzed with
a FACSCaliburÔ cytometer using CELLQuestÔ research software (BD
Biosciences).
4.2.3. Ex vivo experiments
4.2.3.1. Generals. Mice. Female BALB/c (H-2d) mice were bred in
IBMC (Strasbourg, France) animal house facilities (French veteri-
nary service agreement E67-482-2). Animal experimentation was
conducted according to the “Principles of Laboratory Animal Care”
and with the approval of the Regional Ethics Committee of Stras-
bourg (CREMEAS, project no. AL/05/08/03/07).
Acknowledgments
Cells and reagents. D1 cells have been described as a MHC class
II-positive growth factor-dependent immature DC, derived from
adult mouse spleen. Fluorescein isothiocyanate (FITC)-conjugated
anti-mouse CD54 (clone 3E2), FITC-conjugated anti-mouse CD40
(clone 3/23), R-phycoerythrin (PE)-conjugated anti-mouse CD80
(clone 16-10A1), R-PE-conjugated anti-mouse CD86 (clone GL1)
and allophycocyanin (APC)-conjugated anti-mouse CD11c (clone
HL3) monoclonal antibodies were purchased from BD Pharmingen
(San Jose, CA). Low endotoxin recombinant mouse granulocyte/
macrophage colony-stimulating factor (rmGM-CSF) was purchased
from USBiological (Swampscott, MA). Lipopolysaccharide (LPS)
from Escherichia coli (strain 0111.B4) and the polymyxin B were
purchased from SigmaeAldrich (Saint-Louis, MO).
This work was supported by a MRE fellowship allocated to JST.
We gratefully acknowledge the financial support from the Uni-
versité de Strasbourg and the Centre National de la Recherche
Scientifique (C.N.R.S.). The English was revised by Angloscribe,
Calvisson, France.
Appendix A. Supplementary information
Supplementary data related to this article can be found online
doi:10.1016/j.ejmech.2012.02.039.
References
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‘purified B cells’ (containing more than 90% B cells as determined by
flow cytometry analysis), were used in further experiments.
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