Simple synthetic toll-like receptor 2 ligands

Article abstract of DOI:10.1016/j.bmcl.2011.07.102

Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune responses against pathogens followed by slow but specific adaptive immune responses. Development of synthetic TLR2 agonists/antagonists would be useful i

Full text of DOI:10.1016/j.bmcl.2011.07.102

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5863–5865
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Simple synthetic toll-like receptor 2 ligands
⇑
Abu-Baker M. Abdel-Aal, Kalifa Al-Isae, Mehfuz Zaman, Istvan Toth
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Stimulation of toll-like receptor 2 (TLR2) by bacterial lipoproteins induces fast non-specific immune
responses against pathogens followed by slow but specific adaptive immune responses. Development
of synthetic TLR2 agonists/antagonists would be useful in the prevention of different infectious and
immunologic disorders. The current study reports synthesis and TLR2 activity of two simple TLR2 ligands,
which feature minimal structural requirement for TLR2 activity (two long lipid chains) and stimulate
agonistic activity at nanomolar concentration.
Received 5 May 2011
Revised 25 July 2011
Accepted 26 July 2011
Available online 2 August 2011
Keywords:
Toll-like receptor 2
Lipopeptide
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Lipoamino acid
Adjuvant
The innate immune system plays a critical role in the detection
of invading pathogens and activation of suitable protective re-
sponses.1,2 Immune cells are equipped with a set of receptors
which rapidly detect conserved microbial products leading to
acute inflammatory responses and initiation of a cascade of adap-
tive immune responses.3–5 For example, several toll-like receptors
(TLR) are involved in the recognition and immune stimulation
caused by bacterial lipids or lipoproteins (TLR 1, 2, and 6), bacterial
liposaccharides (TLR4), and microbial nucleic acids (TLR 3, 7, 8, and
9).6 Due to the involvement of TLR2 in the recognition of bacterial
lipoproteins, much interest is directed towards the development of
synthetic ligands of this receptor. These efforts have contributed to
our understanding of the role of TLR2 in immune responses.
The ability of TLR2 to modulate immune responses is obviously
shown by the powerful adjuvant activity of synthetic di- and tri-
acylated lipopeptides (Fig. 1; Pam2Cys, Pam3Cys respectively).7
The search for new TLR ligands was supported by the success of
monophosphoryl lipid A as an adjuvant in clinical trials, and its
acceptance by regulatory agencies in Europe and in Australia as a
component of various vaccines.8 Numerous investigations showed
that adjuvant and TLR activity of bacterial lipoproteins are greatly
dependent on the lipid moiety.9–14 It was found that the minimal
structural requirement to activate TLR2 is two ester bound fatty
acids of chain length longer than eight carbon atoms.11 The amide
bound fatty acid is of lesser importance. Biological activity data for
lipoproteins modified at the natural amino acid S-(2,3-dihydroxy-
propyl)-L-cysteine showed the importance of the stereochemical
orientation and the thioether group for activity.10,12 Recently,
X-ray crystal structures of TLR2-ligand complexes have been
revealed which provide good information about the TLR2 structure
as well as its binding to Pam3Cys.15,16 The role of the lipid chains in
binding to TLR1-2 dimer was clearly demonstrated where the two
ester palmitoyl groups bind to a hydrophobic pocket in the TLR2
side while the amide palmitoyl chain is inserted into a hydropho-
bic channel in TLR1.
Lipoamino acids are synthetic amino acids with long alkyl side
chains which combine the properties of amino acids (amphoteric
bifunctional moiety) and lipids. This nature makes them easily
accessible building blocks to design and engineer lipopeptides
which mimic natural TLR2 ligands.17 Simple lipid moieties (518
and 6,19 Fig. 2) incorporating two 16 carbon containing chains
are proposed as novel TLR2 ligands. The compounds feature some
Abbreviations: Boc, t-butoxycarbonyl; DMF, N,N-dimethylformamide; HBTU, 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEK 293,
human embryonic kidney 293; THF, tetrahydrofuran; TFA, trifluoroacetic acid; TLR,
toll-like receptor.
⇑
Corresponding author. Tel.: +61 7 3346 9892; fax: +61 7 3365 1688.
E-mail address: i.toth@uq.edu.au (I. Toth).
Figure 1. Structure of synthetic palmitoyl-S-glyceryl cysteine lipopeptides:
Pam2Cys; R = H, Pam3Cys; R = palmitoyl.
0960-894X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.102

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Abu-Baker M. Abdel-Aal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5863–5865
Figure 2. Synthesis of target lipoamino acid-based TLR2 ligands.
TLR2 - Cells
8
7
6
5
4
3
2
1
0
***
TLR2 + Cells
ns
***
*
Figure 3. Data are shown as means SD of three-culture runs in two separate experiments. Variation between groups were analyzed using the two-tailed Student’s t test and
were regarded statistically significant if the p value was <0.05.
similarity to the lipid moiety of other TLR2 ligands; for example,
Pam2Cys and Pam3Cys The proposed compounds have great advan-
tages over Pam2Cys and Pam3Cys such as stability against esterases
enzymes and oxidation, the ease of synthesis, and low cost produc-
tion. We can also engineer the number and length of lipid chains to
optimize the TLR2 activity. Racemic mixture of lipoamino acids
were tested and it has been shown that chirality influences the
reactivity of similar compounds.20 However for preliminary studies
presented here the focus was on the structure–activity of simple li-
pid moieties with two 16 carbon containing chains. Lipid moieties
such as lipoamino acids can also induce apoptosis of cells.21 As
such a model cell line that is routinely used to verify activity of
similar lipid moieties was used for our compounds.11
an activated palmitic acid. Purification of the benzyl ester by col-
umn chromatography was essential as the presence of any residual
benzyl chloride can lead to side products in the next reaction step.
Deprotection of amino group of compound 2 was accomplished
quantitatively using trifluoroacetic acid to afford benzyl ester 3.
To obtain compound 4, coupling of the activated [HBTU/DIPEA]
palmitic acid to lipoamine 3 was difficult. Using THF as a solvent
led to very low yield. The reaction in DMF increased the yield
and the formation of side products was greatly diminished. Silica
flash column chromatography was applied for purification and
after many attempts it was concluded that using pure DCM eluent
achieved the highest purity with acceptable yield of compound 4
(35%).
The N-Boc-protected 2-aminohexadecanoic lipoamino acid
(Fig. 2, 1) was synthesized as described by Gibbons et al.22 followed
by an O-benzylation reaction. Benzylation was performed to
protect the carboxylic group during the subsequent coupling of
Two subsequent reduction reactions were performed to obtain
target compounds 5 and 6 respectively in high yield (>90%). First
reduction was achieved by hydrogenolysis of benzyl ester 4 in
THF/methanol over palladium as a catalyst to afford acid 5. The

Abu-Baker M. Abdel-Aal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5863–5865
5865
second reaction was carried out in THF with HBTU/DIPEA activa-
References and notes
tion and NaBH4 reduction. The product 6 was purified using silica
gel chromatography (DCM/ether, 4:1).
Human embryonic kidney (HEK293) cells stably expressing
TLR2 were used in TLR2 stimulation experiment. HEK293 cells
were transiently transfected with pNF-jB-Luc Cis-reporter plasmid
as a reporter gene. Negative control wells were treated with media
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analogue. Luciferase activity (NF-jB activation) of the compounds
was measured, normalized against protein concentration and ex-
pressed relative to cells treated with media. Compounds 5 and 6
were tested at 10, 50, and 100 nM concentration (Fig. 3) and the re-
sults indicated that the compounds stimulated signaling through
TLR2 at 100 nM concentration. These results also showed that
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linkage, S-(2,3-dihydroxypropyl)-L-cysteine, the ester functional-
ity] were non-essential for TLR2 activity. Additionally, reduction
of the terminal carboxyl group of compound 5 to give to alcohol
6 did not affect TLR2 activity of the constructs.
TLR 2 recognizes lipoproteins of the outer bacterial membranes.
Beside native lipoproteins, synthetic bacterial lipids such as
Pam2Cys and Pam3Cys also activated TLR2. In an on-going research
to develop a TLR2-activating lipopeptides, we developed lipoamino
acid-based Pam2Cys-like analogues. Two compounds were able to
activate TLR2 receptors at nanomolar level. The proposed
compounds are simpler than other reported synthetic analogues
of bacterial lipoproteins.
Acknowledgment
22. Gibbons, W. A.; Hughes, R. A.; Charalambous, M.; Christodoulou, M.; Szeto, A.;
Aulabaugh, A. E.; Mascagni, P.; Toth, I. Liebigs Ann. Chem. 1990, 1175.
This work was supported by the National Health and Medical
Research Council Australia (NHMRC 496600).