Design, synthesis and biological evaluation of immunostimulating mannosylated desmuramyl peptides

Article abstract of DOI:10.3762/bjoc.15.174

Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adamantane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest enhancement of IgG production was stimulated by compound 21 (Man-OCH₂-(1-Ad)Gly-Ala-isoGln).

Full text of DOI:10.3762/bjoc.15.174

Design, synthesis and biological evaluation of
immunostimulating mannosylated desmuramyl peptides
Rosana Ribić*1, Ranko Stojković2, Lidija Milković2, Mariastefania Antica2, Marko Cigler3
and Srđanka Tomić4
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 1805–1814.
1University Center Varaždin, University North, Jurja Križanića 31b,
HR-42000 Varaždin, Croatia, 2Ruđer Bošković Institute, Bijenička
cesta 54, HR-10000 Zagreb, Croatia, 3Department of Chemistry,
Technical University Munich, Lichtenbergstraße 4, D-85748 Garching,
Germany and 4Faculty of Science, University of Zagreb, Horvatovac
102a, HR-10000 Zagreb, Croatia
doi:10.3762/bjoc.15.174
Received: 16 April 2019
Accepted: 11 July 2019
Published: 29 July 2019
Associate Editor: K. N. Allen
Email:
Rosana Ribić* - rosana.ribic@unin.hr
© 2019 Ribić et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
adamantane; adjuvant activity; desmuramyl peptide; mannose
Abstract
Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl
moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved
pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by
mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel
mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or
C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the
mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adaman-
tane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest
enhancement of IgG production was stimulated by compound 21 (Man-OCH2-ᴅ-(1-Ad)Gly-ʟ-Ala-ᴅ-isoGln).
Introduction
Dendritic cells capture and internalize invading pathogens. down products of polymeric peptidoglycan are called muropep-
Pathogen-associated molecular patterns have been known for a tides. Muropeptides act as agonists of pathogen recognition re-
long time to affect the immune system of mammalian hosts and ceptors (PRRs) and therefore stimulate immune response and
therefore have been extensively studied as possible adjuvants induce T cell differentiation [1-3]. They activate innate immune
for vaccines. Peptidoglycan is a polymeric component of the responses and contribute to the development of adaptive immu-
Gram-positive and Gram-negative bacterial cell wall. Break- nity. Immune response is initiated by the activation of PRRs lo-
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cated on the immune cell surface, by cytosolic or endosomal MDP analogues lacking the N-acetylmuramyl group are called
PRRs. PRRs are classified into: Toll-like receptors (TLRs), desmuramyl peptides. Structure–activity studies of the MDP de-
RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and rivatives and analogues suggest that the ʟ-Ala-ᴅ-isoGln pharma-
C-type lectin receptors (CLRs) [4]. Muramyl dipeptide (MDP, cophore is essential for the immunostimulatory properties but
N-acetylmuramyl-ʟ-alanyl-ᴅ-isoglutamine) is the smallest pepti- the introduction of lipophilic substituent into MDP analogues
doglycan fragment (Figure 1) capable of replacing the whole can increase its adjuvant activity [12,18]. Up to now, our
Mycobacterium in complete Freund’s adjuvant. MDP triggers research was directed towards isomeric desmuramyl peptides
an immune response by activating the mammalian NOD-like re- containing lipophilic unnatural amino acid, adamantylglycine
ceptor, nucleotide binding oligomerization domain-containing (AdGly), bound to the N-terminus of ʟ-Ala-ᴅ-isoGln dipeptide
protein 2 (NOD2). NOD2 is an intracellular protein that signals part as well as their mannosylated derivatives. Different isomers
via the NF-κB pathway to proximally activate innate immunity of mannosylated adamantyl tripeptides, regarding the chiral
through macrophage response as well as to more distally affect centers introduced at adamantylglycine and spacer which
adaptive immunity through the production of antigen-specific T connect the sugar part to the adamantyltripeptide were synthe-
cells [5]. MDP binding to NOD2 has been confirmed [6] sized and biologically evaluated [19-21]. The best adjuvant ac-
as well as the crystal structure of NOD2 in the inactive ADP- tivity in experiments in vivo showed the ManAdTP derivative
bound state [7]. MDP is the structural fragment of the peptido- (Figure 2) which has a ᴅ-configuration at the (adamant-1-
glycan monomer (PGM, Figure 1) which is used in this work. yl)glycine moiety and (R)-configuration at the hydroxyisobu-
PGM is a well-defined and characterized disaccharide tyryl linker. Its activity was higher than PGM that was used as
pentapeptide, β-ᴅ-GlcNAc-(1→4)-ᴅ-MurNAc-ʟ-Ala-ᴅ-isoGln- reference compound.
mesoDAP(εNH2)-ᴅ-Ala-ᴅ-Ala, originating from Brevibac-
terium divaricatum [8,9].
Peptidoglycans activate macrophages via TLR2 receptor,
whereas MDP lacks TLR2-agonistic activity [10]. PGM and
MDP have similar immunostimulating activity and they are
reduced in comparison to the potent complete Freund’s adju-
vant which is used as golden standard for adjuvant activity [11].
However, strong toxicity of complete Freund’s adjuvant
disables its clinical application. MDP is too pyrogenic for clini-
Figure 2: Immunostimulating mannosylated desmuramyl peptide
(ManAdTP).
cal application as well and suffers from rapid elimination.
Therefore, numerous MDP analogues and derivatives were syn-
thesized, in order to improve the properties of the parent mole-
cule [12-16]. Replacement of the N-acetylmuramyl moiety with The results indicate that introduction of mannose plays a signif-
various acyl groups represents an important approach in the icant role in stimulation of the immune response and the possi-
design of new immunologically active MDP analogues [17]. bility of effecting the immune response by the mannose recep-
Figure 1: Peptidoglycan fragments with immunostimulating properties.
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tor family, group I CLRs, present on immunocompetent cells logues based on the ManAdTP hit compound. The attachment
(such as macrophages and dendritic cells) [22,23]. It has been of mannose to the desmuramyl dipeptide may contribute to the
shown that uptake of liposomes displaying mannose ligands at- recognition of the adjuvant compound by specific mannose re-
tached to the surface enhanced the uptake in human monocyte- ceptors expressed at immune cells. Mannosylated drug delivery
derived dendritic cells [24].
systems enhance uptake and activation of dendritic cells and
increase T cell proliferation [24,27,28]. Mannosylation of the
Here we describe a structure–activity relationship (SAR) study adamantylated desmuramyl peptides amplified the adjuvant ac-
on novel mannosylated desmuramyl peptide derivatives in tivity in experiments in vivo [20,21]. Two series of manno-
which two series of compounds were prepared: (i) derivatives conjugates were prepared: (i) derivatives with glycolyl linker
containing a glycolyl linker between mannose and dipeptide, and (ii) the ones with (R)-hydroxyisobutyryl linker which is
and (ii) derivatives containing a parent (R)-hydroxyisobutyryl present in the parent ManAdTP. The glycolic linker was intro-
linker (Figure 3). In both series, positions of adamantane duced due to the fact that N-glycolyl muramyl peptides induce
binding (to the N- or C-terminus) were altered in comparison significantly higher activation of NOD2 than MDP [29,30].
with derivatives lacking the adamantane moiety. Immunostimu- Mycobacteria present in complete Freund’s adjuvant, and
lating properties of synthesized derivatives were assessed in related Actinomycetes, produce N-glycolyl MDP by the
vivo using ovalbumin as an antigen.
hydroxylase action on MDP (N-acetylmuramic acid within the
peptidoglycan). The influence of structural modifications on
immunomodulating properties was estimated by the immunos-
timulatory effect on secondary humoral response to ovalbumin
Results and Discussion
Design
Desmuramyl peptides enter into the cell by passive absorption (antigen) in BALB/c mice.
and this process depends on lipophilicity [25]. Numerous ana-
logues and derivatives which incorporated different lipophilic Chemistry
groups have shown improved activity [14,18,21,26]. To enable Peptide building blocks were prepared starting from the fully
the systematic investigation of influence of lipophilic adaman- protected desmuramyl peptide, Boc-ʟ-Ala-ᴅ-isoGlnOBn. For
tane on the immunostimulating activity, derivatives with altered the synthesis of derivatives lacking the adamantane moiety,
position of the adamantane moiety (at the N- or C-terminus of dipeptide 1 with benzyl protection on the C-terminus was ob-
the dipeptide), as well as derivatives lacking the adamantane tained after Boc-deprotection as previously reported (Scheme 1)
moiety, were prepared. Furthermore, herein we describe a [31]. Peptide 1 was also used for the synthesis of adamantly-
strategy for the preparation of two series of mannose MDP ana- containing desmuramyl tripeptide 3.
Figure 3: General structure of: a) glycolyl and b) (R)-hydroxyisobutyryl derivatives.
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Scheme 1: Synthesis of desmuramyl peptides modified at N-terminus. Reagents and conditions: a) TFA/DCM 1:2, rt, 1 h, quantitative; b) EDC·HCl,
HOBt·H2O, Et3N, DCM/dioxane 1:1, 0 °C → rt, 48 h, 82%; c) TFA/DCM 1:2, rt, 1 h followed by chromatographic separation of isomers, 43%.
Compound 1 was coupled with previously prepared racemic of mannoconjugates. The mannose precursor containing the
Boc-protected (adamant-1-yl)glycine [32] 2 using the carbo- glycolyl linker 11 was prepared in a three-step procedure shown
diimide EDC/HOBt method [21]. The obtained mixture of in Scheme 3.
BocAdGly-ʟ-Ala-ᴅ-isoGlnOBn diastereoisomers was treated
with trifluoroacetic acid in order to remove the Boc protecting The stereoselective α-anomeric deacetylation of peracetylated
group while the diastereoisomer 4 with ᴅ-ʟ-ᴅ amino acid se- mannose 8 was performed using the mild acidic catalyst, zinc
quence was separated from the isomer mixture using silica gel acetate hydrate [33]. The reaction progress was monitored by
column chromatography and CHCl3/MeOH 1:1 as eluent. The thin-layer chromatography. Within the first 20 hours compound
spectral data of the isolated isomer 4 were compared to the 9 was the only product and further progress resulted in gradual
published ᴅ-AdGly-ʟ-Ala-ᴅ-isoGln prepared from tripeptide removal of the remaining acetate groups. The SN2 substitution
tert-butyl ester [21]. Desmuramyl peptides 1 and 4 were further of bromine from tert-butyl bromoacetate with 2,3,4,6-tetra-O-
used for condensation reactions with hydroxyisobutyryl and acetyl-α-ᴅ-mannopyranose (9) in the presence of potassium
glycolyl mannosides. The synthesis of desmuramyl peptide 7 carbonate followed. Chemoselective removal of the tert-butyl
with an adamantane moiety bound at C-terminus is presented in ester group from compound 10 resulted with O-mannoside 11
Scheme 2.
with a free carboxy group available for coupling of the peptide
moieties. The tert-butyl deprotection was accomplished using a
After hydrogenolysis of the starting dipeptide, condensation of selective reagent, trifluoroacetic acid (TFA). Side products
free carboxyl group with adamant-1-ylamine hydrochloride was derived from deacetylation of compound 10 have not been
performed. Boc deprotection of obtained compound 6 gave the detected. The synthesis of benzyl-protected α-mannoside con-
trifluoroacetic salt of peptide 7 which was used in the synthesis taining a (R)-hydroxyisobutyryl linker was previously de-
Scheme 2: Synthesis of C-modified desmuramyl peptides. Reagents and conditions: a) H2, 10% Pd/C, MeOH, 38 psi, rt, 24 h, 96%; b) adamant-1-
ylamine hydrochloride, EDC·HCl, HOBt·H2O, Et3N, DCM/dioxane 1:1, 0 °C → rt, 48 h, 60%; c) TFA/DCM 1:2, rt, 1 h, quantitative.
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Scheme 3: Synthesis of the mannose precursor. Reagents and conditions: a) Zn(OAc)2·H2O, abs. MeOH, rt, 20 h, 60%; b) BrCH2COOC(CH3)3,
K2CO3, dry DMF, rt, 2 h, 81%; c) TFA, dry DCM, rt, 1,5 h, 72%.
scribed [34]. Condensations of peptides 1, 4 and 7 with of MDP?, (ii) which relative position of the adamantane group
carboxy-functionalized mannosides containing a (R)-hydroxy- in the mannosylated desmuramyl peptides causes the greatest
isobutyryl and glycolyl linker are shown in Scheme 4 and increase of the adjuvant activity?
Scheme 5, respectively.
Testing of immunostimulating activity
For the amide bond formation between mannose and peptide The adjuvant activity was estimated by the immunostimulatory
part, an optimized EDC/HOBt method was used in each case. effect on secondary humoral response to the well-established
Synthesized compounds represent a small series of manno- model antigen ovalbumin (OVA) in BALB/c mice (Figure 4)
sylated desmuramyl peptides designed in a way that the intro- according to previously described in vivo studies [11,35]. Anti-
duced structural differences could answer two key questions: OVA IgG, anti-OVA IgG1 and anti-OVA IgG2a were deter-
(i) Will the glycolyl linker in this class of compounds amplify mined in the mice sera after supplementing the mice with the
immunostimulatory activity similar to the N-glycolyl derivative second booster. The comparison of induced anti-OVA IgG
Scheme 4: Synthesis of mannosylated peptides with hydroxyisobutyryl linker. Reagents and conditions: a) EDC·HCl, HOBt·H2O, Et3N, DCM/dioxane
1:1, 0 °C → rt, 48 h, 52–90%; (b) H2, 10% Pd/C, 48 h, rt, 83–92%.
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Scheme 5: Synthesis of mannosylated peptides with glycolyl linker. Reagents and conditions: a) EDC·HCl, HOBt·H2O, Et3N, DCM/DMF 1:1,
0 °C → rt, 72 h, 26–67%; b) NaOMe/MeOH, rt, 1 h, 59–89%.
levels was carried out quantitatively and the subclasses of IgG,
IgG1 and IgG2a, as indicators of Th1 or Th2 type of immune
response, were also determined. The adjuvant activity of syn-
thesized compounds was evaluated in comparison to the
mannosylated adamantyl tripeptide ManAdTP and PGM. PGM
was used as reference adjuvant in previously published studies
since PGM and MDP have similar immunological properties:
both stimulate the Th2-biased immune response specific for the
OVA antigen [11,35].
In general, when compared to the group treated with no adju-
vant (OVA alone), enhancement in total anti-OVA IgG anti-
body production was observed in all groups except in group that
received 16 (Figure 4). High levels of IgG antibody present
even in the OVA treated group, led to a relatively weak stimula-
tion of the total antibody production in the PGM-injected group.
ManAdTP elicited a better immune response than OVA alone
and the PGM-injected group. These results are in very good
agreement with previous research where stimulation of anti-
OVA IgG antibody production by ManAdTP and parent non-
Figure 4: The effect of mannosyl desmuramyl peptides on production
of anti-OVA IgG in BALB/c mice immunized with OVA as an antigen.
Bar graphs represent average values from individual mice from each
group (n = 5). *p < 0.05, **p < 0.01 and ***p < 0.001 denote the statis-
tical significance in comparison to the control group or groups
connected with a line.
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mannosylated AdTP were investigated in the NIH/OlaHsd 16 and 22, respectively. This leads to the conclusion that the
mouse model in comparison to PGM [36]. In BALB/c mice most suitable position of adamantane in this class of com-
used in this study, enhancement in the total anti-OVA IgG anti- pounds is at the peptide N-terminus. Adamantane can act as
body production by ManAdTP is statistically significant membrane anchor for mannose structures and thus be exposed
(p < 0.01). A statistically significant boost in antibody on liposome surfaces and as such used in targeted drug delivery
responses to the OVA antigen was observed in all groups [37]. It can be also incorporated into a β-cyclodextrine cavity, a
immunized with compounds containing a glycolyl linker powerful supramolecular nanoparticle carrier for targeted drug
(p < 0.05 and and p < 0.001) in comparison to the control delivery [38]. Previous research suggested a design of manno-
group. Immunization with compound 21 which has an sylated desmuramyl peptides with adamantane at the
adamantly tripeptide moiety attached to mannose through the C-terminus in order to facilitate the incorporation into the
glycolyl linker led to the highest and statistically most signifi- hydrophobic layer of the cavity because of the minor steric
cant increase in the specific IgG response (p < 0.001). Addition- hindrance of the mannose and peptide part during the inclusion
ally, amplification in the total IgG antibody production was ob- process of the adamantane [37,39].
served in all groups immunized with mannosylated desmu-
ramyl peptides containing a glycolyl linker 20–22 relatively to It is well known that vaccine adjuvants can enhance or modu-
analogues 15, ManAdTP and 16. The results directly indicate late the Th1/Th2-bias of an induced immune response. Inter-
that the introduction of the glycolyl linker plays a significant feron-γ (as a Th1 cytokine) and IL-4 (as a Th2 cytokine) in-
role in stimulation of the immune response in this class of adju- duce isotype switching to IgG2a and IgG1, respectively. There-
vants. Similarly, N-glycolyl muramyl peptides obtained by the fore, isotype profile of antigen specific anti-OVA IgG anti-
oxidation of the N-acetyl group of MDP induce significantly a bodies, IgG1 and IgG2a, is usually measured as a marker of the
higher activation of NOD2 than MDP [29,30]. Glycopeptide 21 Th1 and Th2 type immune response bias [40,41]. In this study,
was identified as the most potent adjuvant in this experiment the type of generated immune response was indirectly esti-
and in this class of adjuvants, so far. Introduction of the mated by quantification of OVA-specific IgG1 (for activation
lipophilic moiety positively influences the adjuvant properties of Th2 type) and IgG2a (for activation of Th1 type) and calcula-
of MDP derivatives and analogues [12]. In both, hydroxyisobu- tion of the respective IgG1/IgG2a ratio. When the amount of
tyryl and glycolyl derivatives, introduction of the bulky and anti-OVA IgG1 antibodies was measured (Figure 5a), it was ob-
lipophilic adamantane showed to be suitable for the immunos- served that in all groups high levels of IgG1 antibody was
timulatory activity. The adjuvant activity changes in respect to present and the highest response, which was also statistically
the position of adamant-1-yl moiety in the peptide part. Anti- significant (p < 0.001), was given by compound 21. A slight
OVA IgG antibody stimulation was higher in the groups immu- suppression was noticed only in the production of anti-OVA
nized with ManAdTP and 21 in respect to groups treated with IgG1 antibodies when compound 16 was administered.
Figure 5: The effect of mannosylated desmuramyl peptides on production of anti-OVA IgG subtypes, anti-OVA IgG1 (a) and anti-OVA IgG2a (b), re-
spectively, in BALB/c mice immunized with OVA as an antigen. Bar graphs represent average values from individual mice from each group (n = 5).
*p < 0.05 and ***p < 0.001 denote statistical significance in comparison to the control group.
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Beilstein J. Org. Chem. 2019, 15, 1805–1814.
A statistically significant enhancement in anti-OVA IgG2a pro- Mannose receptors are one group in the CLRs family which
duction (Figure 5b) was observed in groups immunized with exist as soluble and transmembrane receptors [4]. Like TLRs
mannosylated adamantyl-tripeptides, ManAdTP and 21. The they initiate innate immune responses and activate acquired
type of immune response was indirectly determined by quantifi- immunity. Mannose structures on the other hand, are one of the
cation of IgG1 (for activation of Th2-type) and IgG2a (for acti- glycan structures that build up tumor antigens and regulate the
vation of Th1-type) antibody for each serum (obtained after the immune reaction by specific binding to CLRs. Therefore, com-
second booster) and calculation of the IgG1/IgG2a ratio pounds with expressed CRL agonist or antagonist properties
(Figure 6).
could also be considered as potential agents for cancer
immunotherapy [49,50]. Mannosylated liposomes with incorpo-
rated MDP have proved to be effective carriers for target inhibi-
tion of liver metastasis [51]. Therefore, the presented manno-
sylated desmuramyl peptides with incorporated adamantane
will be further explored in order to get a better insight into
possible PRR crosstalk. Namely, inclusion of adamantane into
carriers such as liposomes can additionally affect the Th1/Th2
switch of the immune response [52]. The presented results
demonstrate the great immunostimulating potential of glycolyl-
modified desmuramyl peptides. Peptidoglycan fragments with
mycobacterial structural features, such as synthesized com-
pounds 20–22, could efficiently link innate and adaptive immu-
nity, similarly as N-glycolyl MDP enhances the innate immune
response and T cell-mediated immunity [53]. NOD2-activation
and interaction with CLR should be further explored, as well as
a potential for synergistic multi-PRR activation.
Conclusion
Figure 6: The ratio of anti-OVA IgG1 and anti-OVA IgG2a (IgG1/
IgG2a) in BALB/c mice. For each mouse serum IgG1/IgG2a was calcu-
lated and the result for each experimental group (n = 5) is presented
as average standard deviation (SD). ***p < 0.001 denote statistical
significance in comparison to the control group.
A series of novel mannosylated desmuramyl peptides were pre-
pared and characterized. In their structures, all glycopeptides
comprised of mannose and the key pharmacophore – desmu-
ramyl peptide. These moieties are connected through a glycolyl
or hydroxyisobutyryl linker and additionally modified on N/C
From the IgG1/IgG2a ratio it is evident that all groups treated terminus with an adamantane subunit. The immunostimulating
with tested adjuvants have higher values than the group treated activities of tested compounds were compared to hit com-
with OVA alone, indicating the slight shift toward more pro- pounds ManAdTP and PGM. In in vivo experiments, all
nounced Th2 type of immune response. Compound 22 signifi- mannopeptides with a glycolyl linker exhibited higher adjuvant
cantly switches immune response toward pronounced Th2 type, activity than analogues with a hydroxyisobutyryl linker indicat-
due to the predominant appearance of IgG1 antibodies. MDP ing that the introduction of the glycolyl moiety plays a signifi-
and PGM dominantly induce the IgG1 antibody production and cant role in the stimulation of the immune response. In particu-
stimulate the Th2-polarized immune response as well [11,42]. It lar, compound 21 was identified, so far, as the most potent adju-
is well known that muropeptides act as NOD2 agonists and in- vant in this class of mannosylated desmuramyl peptides. It
duce a predominant Th2-biased response [43]. NOD2 agonists should be also noted that the compound 21 is stable, non-pyro-
have the ability to act synergistically and augment the adjuvant genic and water-soluble what makes it potentially applicable as
activity of TLR ligands [10,44-46]. Furthermore, a combina- an adjuvant for vaccines.
tion of PRR ligands, such as NLR/TLR, induce Th1-polarized
response. This NLR/TLR crosstalk is essential for modulation
of innate and adaptive immune responses and leads to develop-
Supporting Information
ment of new approaches for the design of novel vaccines. Ap-
Supporting Information File 1
plication of multi-PRR activation approaches can increase the
immunity significantly [47]. Another example of dual adjuvant
system is represented by the activation of dendritic cells via
combined macrophage-inducible CLR and TLR ligands [48].
Experimental and characterization data.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-174-S1.pdf]
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Beilstein J. Org. Chem. 2019, 15, 1805–1814.
18.Khan, F.-A.; Ulanova, M.; Bai, B.; Yalamati, D.; Jiang, Z.-H.
Acknowledgements
Eur. J. Med. Chem. 2017, 141, 26–36.
doi:10.1016/j.ejmech.2017.09.070
This work has been supported by the Croatian Science Founda-
tion under projects IP-2014-09-7899 and IP-11-2013 7140, the
Terry Fox Foundation, CERRM of the University of Zagreb,
School of Medicine funded through European Regional Devel-
opment Fund, Operational Programme Competitiveness and
Cohesion.
19.Vranešić, B.; Tomašić, J.; Smerdel, S.; Kantoci, D.; Benedetti, F.
Helv. Chim. Acta 1993, 76, 1752–1758. doi:10.1002/hlca.19930760431
20.Ribić, R.; Habjanec, L.; Vranešić, B.; Frkanec, R.; Tomić, S.
Croat. Chem. Acta 2011, 84, 233–244. doi:10.5562/cca1827
21.Ribić, R.; Habjanec, L.; Vranešić, B.; Frkanec, R.; Tomić, S.
Chem. Biodiversity 2012, 9, 777–788. doi:10.1002/cbdv.201100232
22.Geijtenbeek, T. B. H.; Gringhuis, S. I. Nat. Rev. Immunol. 2009, 9,
465–479. doi:10.1038/nri2569
ORCID® iDs
Rosana Ribić - https://orcid.org/0000-0001-7611-538X
Lidija Milković - https://orcid.org/0000-0002-4484-039X
Mariastefania Antica - https://orcid.org/0000-0001-8449-1078
Marko Cigler - https://orcid.org/0000-0003-1861-7968
23.Gazi, U.; Martinez-Pomares, L. Immunobiology 2009, 214, 554–561.
doi:10.1016/j.imbio.2008.11.004
24.White, K. L.; Rades, T.; Furneaux, R. H.; Tyler, P. C.; Hook, S.
J. Pharm. Pharmacol. 2006, 58, 729–737. doi:10.1211/jpp.58.6.0003
25.Smrdel, P.; Grabnar, I.; Locatelli, I.; Černe, M.; Andrenšek, S.;
Kovačič, N.; Kristl, A.; Bogataj, M.; Urleb, U.; Mrhar, A.
Drug Dev. Ind. Pharm. 2009, 35, 1293–1304.
Preprint
A non-peer-reviewed version of this article has been previously published
as a preprint doi:10.3762/bxiv.2019.8.v1
doi:10.3109/03639040902889814
26.Willems, M. M. J. H. P.; Zom, G. G.; Meeuwenoord, N.; Khan, S.;
Ossendorp, F.; Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V.;
Codée, J. D. C. ChemMedChem 2016, 11, 190–198.
References
doi:10.1002/cmdc.201500196
1. Awate, S.; Babiuk, L. A.; Mutwiri, G. Front. Immunol. 2013, 4, No. 114.
doi:10.3389/fimmu.2013.00114
27.Yang, R.; Xu, J.; Xu, L.; Sun, X.; Chen, Q.; Zhao, Y.; Peng, R.; Liu, Z.
ACS Nano 2018, 12, 5121–5129. doi:10.1021/acsnano.7b09041
28.Kramer, S.; Langhanki, J.; Krumb, M.; Opatz, T.; Bros, M.; Zentel, R.
Macromol. Biosci. 2019, 19, 1800481. doi:10.1002/mabi.201800481
29.Raymond, J. B.; Mahapatra, S.; Crick, D. C.; Pavelka, M. S., Jr.
J. Biol. Chem. 2005, 280, 326–333. doi:10.1074/jbc.m411006200
30.Chen, K.-T.; Huang, D.-Y.; Chiu, C.-H.; Lin, W.-W.; Liang, P.-H.;
Cheng, W.-C. Chem. – Eur. J. 2015, 21, 11984–11988.
doi:10.1002/chem.201501557
2. Nielsen, A. E.; Hantho, J. D.; Mancini, R. J. Future Med. Chem. 2017,
9, 1345–1360. doi:10.4155/fmc-2017-0101
3. Traub, S.; von Aulock, S.; Hartung, T.; Hermann, C. J. Endotoxin Res.
2006, 12, 69–85. doi:10.1177/09680519060120020301
4. Kingeter, L. M.; Lin, X. Cell. Mol. Immunol. 2012, 9, 105–112.
doi:10.1038/cmi.2011.58
5. Ogawa, C.; Liu, Y.-J.; Kobayashi, K. S. Curr. Bioact. Compd. 2011, 7,
180–197. doi:10.2174/157340711796817913
31.Ribić, R.; Kodrin, I.; Biljan, I.; Paurević, M.; Tomić, S. Struct. Chem.
2019, 30, 743–754. doi:10.1007/s11224-018-1234-x
6. Lauro, M. L.; D’Ambrosio, E. A.; Bahnson, B. J.; Grimes, C. L.
ACS Infect. Dis. 2017, 3, 264–270. doi:10.1021/acsinfecdis.6b00154
7. Maekawa, S.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T.
Nat. Commun. 2016, 7, 11813. doi:10.1038/ncomms11813
8. Keglević, D.; Ladešić, B.; Tomašić, J.; Valinger, Z.; Naumski, R.
Biochim. Biophys. Acta, Gen. Subj. 1979, 585, 273–281.
doi:10.1016/0304-4165(79)90027-8
9. Halassy, B.; Krstanović, M.; Frkanec, R.; Tomašić, J. Vaccine 2003,
21, 971–976. doi:10.1016/s0264-410x(02)00547-9
10.Tada, H.; Aiba, S.; Shibata, K.-I.; Ohteki, T.; Takada, H. Infect. Immun.
2005, 73, 7967–7976. doi:10.1128/iai.73.12.7967-7976.2005
11.Habjanec, L.; Halassy, B.; Tomašić, J. Int. Immunopharmacol. 2010,
10, 751–759. doi:10.1016/j.intimp.2010.04.005
32.Clariana, J.; Garcıa-Granda, S.; Gotor, V.; Gutiérrez-Fernández, A.;
Luna, A.; Moreno-Mañas*, M.; Vallribera, A. Tetrahedron: Asymmetry
2000, 11, 4549–4557. doi:10.1016/s0957-4166(00)00428-6
33.Kaya, E.; Sonmez, F.; Kucukislamoglu, M.; Nebioglu, M. Chem. Pap.
2012, 66, 312–315. doi:10.2478/s11696-012-0143-5
34.Ribić, R.; Kovačević, M.; Petrović-Peroković, V.; Gruić-Sovulj, I.;
Rapić, V.; Tomić, S. Croat. Chem. Acta 2010, 83, 421–431.
35.Tomašić, J.; Hanzl-Dujmović, I.; Špoljar, B.; Vranešić, B.; Šantak, M.;
Jovičić, A. Vaccine 2000, 18, 1236–1243.
doi:10.1016/s0264-410x(99)00382-5
36.Ribić, R.; Habjanec, L.; Frkanec, R.; Vranešić, B.; Tomić, S.
Chem. Biodiversity 2012, 9, 1373–1381. doi:10.1002/cbdv.201200008
37.Štimac, A.; Šegota, S.; Dutour Sikirić, M.; Ribić, R.; Frkanec, L.;
Svetličić, V.; Tomić, S.; Vranešić, B.; Frkanec, R.
12.Rubino, S. J.; Magalhaes, J. G.; Philpott, D.; Bahr, G. M.; Blanot, D.;
Girardin, S. E. Innate Immun. 2013, 19, 493–503.
doi:10.1177/1753425912471691
Biochim. Biophys. Acta, Biomembr. 2012, 1818, 2252–2259.
doi:10.1016/j.bbamem.2012.04.002
13.Gobec, M.; Tomašič, T.; Štimac, A.; Frkanec, R.; Trontelj, J.;
Anderluh, M.; Mlinarič-Raščan, I.; Jakopin, Ž. J. Med. Chem. 2018, 61,
2707–2724. doi:10.1021/acs.jmedchem.7b01052
14.Gobec, M.; Mlinarič-Raščan, I.; Sollner Dolenc, M.; Jakopin, Ž.
Eur. J. Med. Chem. 2016, 116, 1–12.
doi:10.1016/j.ejmech.2016.03.030
15.Meyers, P. A. Expert Rev. Anticancer Ther. 2009, 9, 1035–1049.
doi:10.1586/era.09.69
38.Štimac, A.; Šekutor, M.; Mlinarić-Majerski, K.; Frkanec, L.; Frkanec, R.
Molecules 2017, 22, 297. doi:10.3390/molecules22020297
39.Car, Ž.; Kodrin, I.; Požar, J.; Ribić, R.; Kovačević, D.; Peroković, V. P.
Tetrahedron 2013, 69, 8051–8063. doi:10.1016/j.tet.2013.06.097
40.Yip, H. C.; Karulin, A. Y.; Tary-Lehmann, M.; Hesse, M. D.; Radeke, H.;
Heeger, P. S.; Trezza, R. P.; Heinzel, F. P.; Forsthuber, T.;
Lehmann, P. V. J. Immunol. 1999, 162, 3942–3949.
41.Ioannou, X. P.; Gomis, S. M.; Karvonen, B.; Hecker, R.; Babiuk, L. A.;
van Drunen Littel-van den Hurk, S. Vaccine 2002, 21, 127–137.
doi:10.1016/s0264-410x(02)00378-x
16.Dzierzbicka, K.; Kolodziejezyk, A. M. Pol. J. Chem. 2003, 77, 373–395.
17.Jakopin, Ž.; Gobec, M.; Mlinarič-Raščan, I.; Sollner Dolenc, M.
J. Med. Chem. 2012, 55, 6478–6488. doi:10.1021/jm300503b
1813

Beilstein J. Org. Chem. 2019, 15, 1805–1814.
42.Magalhaes, J. G.; Fritz, J. H.; Le Bourhis, L.; Sellge, G.;
Travassos, L. H.; Selvanantham, T.; Girardin, S. E.;
Gommerman, J. L.; Philpott, D. J. J. Immunol. 2008, 181, 7925–7935.
doi:10.4049/jimmunol.181.11.7925
43.Girardin, S. E.; Boneca, I. G.; Viala, J.; Chamaillard, M.; Labigne, A.;
Thomas, G.; Philpott, D. J.; Sansonetti, P. J. J. Biol. Chem. 2003, 278,
8869–8872. doi:10.1074/jbc.c200651200
44.Traub, S.; von Aulock, S.; Hartung, T.; Hermann, C. J. Endotoxin Res.
2006, 12, 69–85. doi:10.1179/096805106x89044
45.Tukhvatulin, A. I.; Dzharullaeva, A. S.; Tukhvatulina, N. M.;
Shcheblyakov, D. V.; Shmarov, M. M.; Dolzhikova, I. V.;
Stanhope-Baker, P.; Naroditsky, B. S.; Gudkov, A. V.; Logunov, D. Y.;
Gintsburg, A. L. PLoS One 2016, 11, e0155650.
doi:10.1371/journal.pone.0155650
46.Roychowdhury, A.; Wolfert, M. A.; Boons, G.-J. ChemBioChem 2005,
6, 2088–2097. doi:10.1002/cbic.200500181
47.Tom, J. K.; Albin, T. J.; Manna, S.; Moser, B. A.; Steinhardt, R. C.;
Esser-Kahn, A. P. Trends Biotechnol. 2019, 37, 373–388.
doi:10.1016/j.tibtech.2018.10.004
48.van Haren, S. D.; Dowling, D. J.; Foppen, W.; Christensen, D.;
Andersen, P.; Reed, S. G.; Hershberg, R. M.; Baden, L. R.; Levy, O.
J. Immunol. 2016, 197, 4413–4424. doi:10.4049/jimmunol.1600282
49.Yan, H.; Kamiya, T.; Suabjakyong, P.; Tsuji, N. M. Front. Immunol.
2015, 6, No. 408. doi:10.3389/fimmu.2015.00408
50.Glaffig, M.; Stergiou, N.; Hartmann, S.; Schmitt, E.; Kunz, H.
ChemMedChem 2018, 13, 25–29. doi:10.1002/cmdc.201700646
51.Opanasopit, P.; Sakai, M.; Nishikawa, M.; Kawakami, S.;
Yamashita, F.; Hashida, M. J. Controlled Release 2002, 80, 283–294.
doi:10.1016/s0168-3659(02)00006-8
52.Habjanec, L.; Frkanec, R.; Halassy, B.; Tomašić, J. J. Liposome Res.
2006, 16, 1–16. doi:10.1080/08982100500528537
53.Behr, M. A.; Divangahi, M. Curr. Opin. Microbiol. 2015, 23, 126–132.
doi:10.1016/j.mib.2014.11.015
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