Novel powerful water-soluble lipid immunoadjuvants inducing mouse dendritic cell maturation and B cell proliferation using TLR2 pathway

Article abstract of DOI:10.1016/j.bmcl.2010.01.146

Four novel water-soluble lipid immunoadjuvants were designed, synthesized and characterized by MS and NMR. They all induce mouse dendritic cell maturation and B cell proliferation. We demonstrate that in spite of the chemical modification, the four compou

Full text of DOI:10.1016/j.bmcl.2010.01.146

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1869–1872
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel powerful water-soluble lipid immunoadjuvants inducing mouse
dendritic cell maturation and B cell proliferation using TLR2 pathway
Maria Vittoria Spanedda a, Béatrice Heurtault a, Steffen Weidner a, Corinne Baehr a, Emmanuelle Boeglin b,
a,
Julien Beyrath b, Sara Milosevic b, Line Bourel-Bonnet a, Sylvie Fournel b, , Benoît Frisch
*
*
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 Immunologie et Chimie Thérapeutiques, UPR 9021 CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel water-soluble lipid immunoadjuvants were designed, synthesized and characterized by MS
and NMR. They all induce mouse dendritic cell maturation and B cell proliferation. We demonstrate that
in spite of the chemical modification, the four compounds remain TLR2 agonists.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 14 December 2009
Revised 28 January 2010
Accepted 29 January 2010
Available online 2 February 2010
Keywords:
TLR2 agonist
Water-soluble
Immunoadjuvant
In the present vaccine context, new original immunoadjuvants
are more than ever needed.1–3 In this field, synthetic analogs of tri-
acylated and diacylated lipopeptides derived from the N-terminal
domain of respectively bacterial or mycoplasmal lipoproteins are
highly potent immunoadjuvants when administered either in com-
bination with protein antigens or covalently linked to small anti-
genic peptides.4,5 It is now well established that these molecules
act as agonists on Toll Like Receptors (TLRs) heterodimers and acti-
vate cells of innate and adaptive immune systems as agonists. They
act specially on TLRs 2/1 and 2/6 (for triacylated and diacylated
lipopeptides, respectively).6,7
Despite little is known about the molecular basis of ligand rec-
ognition, one of the key physico-chemical properties of this kind of
compounds is their amphiphilicity. Though it is a cornerstone in
their activity, amphiphilicity settles some practical problems of
solubility that are overcome when the above-mentioned lipoadju-
vants are covalently bound to hydrophilic proteins or peptides
imposing their hydrophilicity on the final product. Amphiphilicity
is also turned as an advantage in vaccine experiments where, for
example, an analogue of ‘Pam3CAG’ (in other words, N-
a-
palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-
L
L
glycine, 1) is incorporated into liposomes. The fatty part of these
synthetic lipopeptides interacts with liposome lipid bilayer
whereas their hydrophilic head is modified covalently to anchor
one or two peptide containing T epitopes against which an im-
mune response is expected.8,9 Nevertheless, if such a formulation
is not desirable, the amphiphilicity is a drawback that hampers
the easy use of lipopeptidic immunoadjuvants both in vitro and
in vivo. Several studies proved that lipopeptides aggregated in
water solutions and that large and heterogeneous aggregates were
responsible for the loss of activity.10 In this context, it has been re-
ported that activity of such lipopeptides was critically dependent
on the dilution protocol (e.g., with dimethylsulfoxide or tert-butyl
alcohol) and on the presence of proteins (fetal bovine serum, bo-
Abbreviations: Boc, tertiobutyloxycarbonyl; BOP, (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate; CD, cluster of differentiation;
DC, dendritic cell; DCC, dicyclohexylcarbodiimide; DIPEA, diisopropylethylamine;
DMAP, dimethylaminopyridine; LPS, lipopolysaccharide; Pam, palmitoyl; Pam3
CAG-OH, N-a-palmitoyl-S-[23-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteinyl-L-ala-
nyl-glycine; PEG, polyethyleneglycol; TFA, trifluoroacetic acid; TLR, toll-like
vine serum albumin. . .) or detergents (octyl-b-D-glucopyranoside)
acting as solubilizers.11
To avoid complicated dissolution/dispersion protocols and the
use of solubilizers, we developed synthetic analogs of ‘Pam3CAG’
(1) that could be more hydrophilic and, if possible, water-soluble.
To increase the water solubility of compounds of interest, various
strategies could be used12 as, for example, the introduction of sup-
plementary charges on the molecule. For this, the addition of prot-
onable functions on lipopeptidic immunoadjuvant thanks to a
receptor.
* Corresponding authors. Tel.: +33 (0)3 88 41 70 24; fax: +33 (0)3 88 61 06 80
(S.F.); tel.: +33 (0)3 68 85 41 68; fax: +33 (0)3 68 85 43 06 (B.F.).
E-mail addresses: s.fournel@unistra.fr (S. Fournel), frisch@unistra.fr, lbourel@
unistra.fr (B. Frisch).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.146

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M. V. Spanedda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1869–1872
polylysine head has already been described and is commercially
available.13 Nevertheless, such an addition reinforces the amphi-
philic character of the analogs, therefore non-specific adhering
properties can be encountered. To avoid the polycationic distur-
bance, we chose to increase the overall hydrophilicity without
modifying the final charges. Therefore, we chose to add a polyeth-
ylene glycol (PEG) chain.14 A lipopeptide-PEG conjugate was previ-
ously evaluated on the lymphocyte B activity and was found to
keep the same properties than the hydrophilic counterpart.15
Moreover, Heucking and co-workers recently reported the charac-
terization of similar compounds modified by a PEG without report-
ing biological results.16 Here we chose the latter strategy to
improve the water solubility of the analogs of ‘Pam3CAG’ and
‘Pam2CAG’ by a PEG2000. These new compounds were evaluated
on mouse dendritic cell maturation, B cell proliferation and proved
to involve the TLR2 pathway.
O
O
H
H
To introduce a PEG linker on N-a-palmitoyl-S-[2,3-bis(palmi-
N
N
R1
R2
N
toyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-glycine (1) and its ana-
L
L
H
O
logs ( Fig. 1), the accurate study of the molecule revealed that
some parts could be modified more easily than others. To find
which ones, we reconsidered the synthetic routes and the struc-
ture/activity relationships previously established.17 Among the
prominent modifications, an amide bond between the C-terminal
function of 1 and the amine of a mono reactive PEG2000 of the type
H2N–(CH2–CH2–O)n–CH2–CH2–OCH3 (where n ꢀ 44) was created
to get compound 2 (Scheme 1). On the synthetic route to 1
S
O
O
O
O
Figure 1. General structure for lipopeptides 1–5. Compound 1: Pam3CAG-OH
R
1 = Pam, R2 = OH. Compound 2: R1 = Pam, R2 = NH–(CH2–CH2–O)n–CH2–CH2–OCH3
(described as TLR2/TLR1 dimer ligand) is the diacylated N-
a-H-S-
(n ꢀ 44). Compound 3: R1 = H (TFA salt), R2 = NH–(CH2–CH2–O)n–CH2–CH2–OCH3
(n ꢀ 44). Compound 4: R1 = CH3O–(CH2–CH2–O)n–CH2–CO (n ꢀ 44), R2 = OH. Com-
pound 5: R1 = H (TFA salt), R2 = NH–(CH2)2–S–S–(CH2)2–NH–CO–CH2–NH–COO–
(CH2–CH2–O)n–CH2–CH2–OH (n ꢀ 44).
[2,3-bis(palmitoyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-glycine
L
L
(improperly called ‘Pam2CAG’, described as TLR2/TLR6 dimer
ligand) where the N-terminal position is still free. In a previous
O
O
H
N
O
O
H
N
PamHN
O
PamHN
N
H
OH
d
N
H
N
H
O
n
O
O
S
S
PamO
PamO
OPam
OPam
2
1
a, b, c
O
O
H
O
H N
N
TFA
2
N
H
N
O
H
n
O
S
c, d, a
a, e, c
PamO
O
O
H
N
OPam
BocHN
S
N
H
OBn
O
3
PamO
OPam
O
O
H
H
O
N
N
N
OH
O
n
H
O
O
S
PamO
OPam
O
H
N
S
OH
n
H2N
S
N
H
O
O
4
c, f, a
O
O
O
H
N
H
N
H2N
S
S
OH
n
TFA
N
N
H
N
H
O
H
O
O
S
PamO
OPam
5
Scheme 1. Synthesis of PEG2000 derivatives. Reagents and conditions: (a) TFA, CH2Cl2, 12 h, rt; (b) PamOH, DCC, DMAP, CH2Cl2, 6 h, rt; (c) H2, Pd/C, CH2Cl2/MeOH, 12 h; (d)
H2NPEG2000OMe, BOP, DIPEA, CH2Cl2, 12 h, rt; (e) MeOPEG2000COOH, BOP, DIPEA, CH2Cl2, 12 h, rt; (f) BOP, DIPEA, CH2Cl2, 12 h, rt.

M. V. Spanedda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1869–1872
1871
work, we demonstrated that this position is quite tolerant in con-
trast to the rest of the molecule, especially the cystein side chain,
that needs to be at least double acylated in order to keep affinity
for the lipid bilayer and immunoadjuvant activity.18–20 Therefore,
both free amine and corresponding amides, in which the amine
was acylated by a non-fatty acid, have been described to remain
active. As a consequence, we designed both compound 3 where
an amide is created between the C-terminal function of ‘Pam2CAG’
Briefly, the two carboxylic acids of (Boc-
L
-Cys-OH)2 were esterified
by benzyl alcohol under phase transfer conditions (water saturated
by NaHCO3, pH 8.5, CH2Cl2 as solvents and tricaprylylmethylam-
monium chloride as phase transfer agent). The SN2 reaction was
performed in 24 h at room temperature within 86% yield. Then, after
a complete reduction by an excess of zinc for 2 h in acidic conditions,
thethiol reacted on (R)-(+)-glycidolfor 36 h at 40 °C within 31% yield
to get a one pot thioether bond. At this step, the separation of unre-
acted (R)-(+)-glycidol from the expected compound was tricky. After
purification, the two alcohols were acylated by palmitic acid after
activation by DCC in the presence of DMAP within a 93% yield. Final-
ly, the benzyl protection was removed under catalytic hydrogena-
tion conditions (high pressure H2 was used over Pd/C in a CH2Cl2/
and the amine of the same mono reactive amino-PEG2000 (N-
a-H-S-
[2,3-bis(palmitoyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-glycinyl-
L
L
NH-PEG2000-OMe) and compound 4 where the N-terminal amine is
acylated by a mono reactive PEG2000 of the type CH3O–(CH2–
CH2–O)n–CH2–COOH (where n ꢀ 44), that is, N-
a-PEG2000-S-
[2,3-bis(palmitoyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-glycine.
L
L
MeOH mixture) to get a first scaffold N-
oxy)-(2R)-propyl]- -cysteine within 88% yield and a 23% overall
yield. The expected scaffold N- -H-S-[2,3-bis(palmitoyloxy)-(2R)-
propyl]- -cysteinyl- -alanyl-glycine was obtained by the conver-
a-Boc-S-[2,3-bis(palmitoyl-
Finally, on the basis of compound 3 (with only two fatty chains),
another analog, in which the PEG2000 chain was linked to a
disulfure bridge labile in proper red/ox conditions was designed.
The rational for this modification lies on the prodrug concept21
L
a
L
L
gent formation of an amide between the C-terminal and an
alanyl-glycyl-derivative properly chosen. Syntheses of compounds
4 and 5 were then performed by peptidic couplings with the corre-
sponding PEG2000 moiety starting from this common scaffold. Rea-
sonable yields were encountered, that is, 2: 25% starting from
‘Pam3CAG’, 3: 31% from ‘BocPam2CAG’, 4: 35% from ‘Pam2CAG-
OBn’ and 5: 27% from ‘BocPam2CAG’. All these compounds display
an interesting solubility profile as they are soluble in dichlorometh-
ane and chloroform as apolar solvents, in methanol but above all, the
four compounds are soluble in water (1 mg/mL).
asserting that the hydrophilicity of compound 5 (N-
a-H-S-[2,3-
bis(palmitoyloxy)-(2R)-propyl]- -cysteinyl- -alanyl-glycinyl-NH–
L
L
(CH2)2–S–S–(CH2)2–NH–CO–CH2–NH–COO–(CH2–CH2–O)n–CH2–
CH2–OH, n ꢀ 44), was necessary for its bioavailability and could
be removed at the expected site by reduction.
Thanks to a convergent strategy, four analogs of the above-
mentioned ‘Pam3CAG’ were thus synthesized (Scheme 1). For every
compound, the first steps were identical until a common ‘Pam2-
Cys-Ala-Gly-OH’ scaffold was obtained (see Supplementary data).
A
CD86
CD54
100
5
2
3
4
LPS
5
2
3
4
LPS
medium
medium
0
Relative fluorescence intensity (4 decade log)
B
4
3
2
5
C
30
25
20
15
10
5
25
CD86
CD54
20
15
10
5
0
0
0
0.2
0.4
0.6
0.8
1
1.2
4
3
2
5
-
-
-
-
0.5
5
-
-
0.5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.5
5
-
-
0.5
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
5
-
-
-
-
[TLR2 agonists] µg/mL
-
-
0.5 5
-
0.5
-
5
-
-
-
-
-
-
0.5 5
-
-
-
-
0.5 5
Figure 2. Water soluble analogs induce mouse dendritic cell maturation and mouse spleen B cell proliferation. (A, B) Maturation of D1 mouse dendritic cell line. Cells (2.105)
were cultured for 48 h before addition of 2, 3, 4 or 5 analogs at 5 g/ml (A, B) or 0.5 g/mL (B). As positive control LPS (5 g/mL) was used. After 24 h, expression of CD54 and
l
l
l
CD86 activation markers was measured by flow cytometry. (A) One representative histogram and (B) MFI (mean fluorescence intensities) measured for each maturation
marker (on experiment representative of 3). (C) Proliferation of mouse spleen B cells. Purified spleen B cells were cultured with the different analogs at the indicated
concentration for 72 h. Proliferation was then evaluated by measuring the [3H]-thymidine uptake. Data are expressed as mean SI SD of three independent experiments
(each experiment was performed in triplicate). SI = stimulation index which is the ratio of the average counts per minute in stimulated cells to the average counts per minute
for unstimulated cells.

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M. V. Spanedda et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1869–1872
20
B
A
70
60
50
40
15
10
5
0
medium
Anti-TLR2
4
3
2
5
+
-
-
-
-
-
-
-
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
medium
+
-
-
-
-
-
-
-
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
+
+
+
Anti-TLR2
+
-
+
+
+
+
-
-
+
+
-
+
-
-
-
+
-
-
-
-
+
-
-
+
+
-
+
-
-
-
+
-
-
-
-
+
4
3
2
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
+
Figure 3. Water soluble analog-induced activation is decreased by antagonist anti-TLR2 antibody DC maturation (A) and B cell proliferation (B) were measured as in Figure 2
except that antagonist anti-TLR2 antibodies (clone HM1054) were added at 1 g/mL 25 min before addition of the various water soluble analogs used at 0.5 g/mL (A) or
0.16 g/mL (B).
l
l
l
To evaluate if the water soluble analogs retain their biological
activity, we checked their ability to induce mouse dendritic cell
maturation22 and mouse spleen B cell proliferation,23 two well de-
scribed biological effects of TLR2 agonists.
Strasbourg, Centre National de la Recherche Scientifique
(C.N.R.S.). J.B. and E.B. were granted by Region Alsace, ARC and
Ministère de la Recherche, respectively.
We investigated the capacity of the analogs to induce DC mat-
uration by using the mouse DC line D124 which matures upon
TLR signaling. As shown in Figure 2A and B, the four analogs in-
duced an increase in the expression of the activation markers
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.146.
CD54 and CD86 at concentration ranging from 0.5 to 5 lg/mL.
Whereas compounds 2, 3 and 5 induced a maturation level similar
to that induced by LPS, compound 4 is less efficient. For note,
experiments using the water soluble analogs were performed in
the presence of the LPS activity inhibitor polymyxin B to rule out
the possibility of contaminating LPS D1 maturation.
References and notes
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Opin. Chem. Biol. 2009, 13, 354.
We next investigated the capacity of the four analogs (2–5) to
induce B cell proliferation. For this, B cells were isolated from
mouse spleen and cultured in the presence of the analogs. Com-
pounds 2–5 induced B cell proliferation (Fig. 2C) from the low con-
centration of 0.02 lg/mL. As for DC maturation, compound 4 was
less efficient in inducing B cell proliferation.
3. Bundle, D. R. Nat. Chem. Biol. 2007, 3, 605.
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Finally, to ensure that water soluble analogs induce DC matura-
tion and B cell proliferation via TLR2 pathway, we repeated the
experiments in the presence of antagonist anti-TLR2 antibody that
inhibits the binding of TLR2 agonist to TLR2. As shown in Figure 3,
the four analog activity was dramatically reduced in the presence
of anti-TLR2 antagonist confirming that the biological activities
of the water soluble analogs involve the TLR2 pathway.
In conclusion, we synthesized some new water soluble lipid
immunoadjuvants. The biological results obtained with our inno-
vative molecules on mouse dendritic cells as well as on mouse B
cell showed that these compounds are still able to induce a strong
immune response. We therefore demonstrated that they involve
the TLR2 pathway. To the best of our knowledge, though ‘Pam3C’
water-soluble analogs were already reported, it is the first time
that ‘Pam2CAG’ water-soluble analogs are described together with
a biological characterization of their immunoadjuvant profile. This
class of molecules will be incorporated in vaccine cocktails for can-
cer immunotherapy or infectious disease prevention, giving the
opportunity to afford safer and more efficient vaccines, a point of
crucial importance in the present health context.
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Acknowledgements
We thank Patrick Wehrung and Pascale Buisine for ESI-MS. We
gratefully acknowledge financial support from Université de