Design, automated synthesis and immunological evaluation of NOD2-ligand-antigen conjugates

Article abstract of DOI:10.3762/bjoc.10.148

The covalent attachment of an innate immune system stimulating agent to an antigen can provide active vaccine modalities capable of eliciting a potent immune response against the incorporated antigen. Here we describe the design, automated synthesis and i

Full text of DOI:10.3762/bjoc.10.148

Design, automated synthesis and immunological
evaluation of NOD2-ligand–antigen conjugates
Marian M. J. H. P. Willems1, Gijs G. Zom2, Nico Meeuwenoord1,
Ferry A. Ossendorp2, Herman S. Overkleeft1, Gijsbert A. van der Marel1,
Jeroen D. C. Codée*1 and Dmitri V. Filippov*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1445–1453.
1Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands and 2Department of
Immunohematology and Blood Transfusion, Leiden University Medical
Centre, P. O. Box 9600, 2300 RC Leiden, The Netherlands
doi:10.3762/bjoc.10.148
Received: 21 February 2014
Accepted: 24 April 2014
Published: 26 June 2014
Email:
Jeroen D. C. Codée* - jcodee@chem.leidenuniv.nl;
Dmitri V. Filippov* - filippov@chem.leidenuniv.nl
Editor-in-Chief: P. H. Seeberger
© 2014 Willems et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
automated synthesis; glycopeptide; innate immunity; muramyl
dipeptide; NOD2 receptor; solid phase synthesis
Abstract
The covalent attachment of an innate immune system stimulating agent to an antigen can provide active vaccine modalities capable
of eliciting a potent immune response against the incorporated antigen. Here we describe the design, automated synthesis and
immunological evaluation of a set of four muramyl dipeptide–peptide antigen conjugates. Muramyl dipeptide (MDP) represents a
well-known ligand for the intracellular NOD2 receptor and our study shows that covalently linking an MDP-moiety to an antigenic
peptide can lead to a construct that is capable of stimulating the NOD2 receptor if the ligand is attached at the anomeric center of
the muramic acid. The constructs can be processed by dendritic cells (DCs) and the conjugation does not adversely affect the
presentation of the incorporated SIINFEKL epitope on MHC-I molecules. However, stimulation of the NOD2 receptor in DCs was
not sufficient to provide a strong immunostimulatory signal.
Introduction
In recent years the study of pattern recognition receptors PRR-ligand (PRR-L) covalently bound to antigenic proteins and
(PRRs) and associated ligands has evolved tremendously [1]. oligopeptides in the development of new (semi)synthetic
The discovery of Toll-like receptors (TLRs) [2] in the late vaccine modalities [3-9]. For instance, the group of Boons
1990s has had a major impact on the field of immunology. This investigated a three-component conjugate containing a tumor-
is reflected in the exploration of conjugates consisting of a associated glycopeptide and a T cell epitope covalently bound
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to a TLR2 ligand [10,11]. Previously we have reported on the N-acetylmuramic acid (MurNAc) of which the lactic acid is
design, synthesis and immunological evaluation of constructs of connected to a peptide [24]. Where NOD1 recognizes
TLR ligands covalently linked to a synthetic long peptide diaminopimelic acid containing peptides [25,26], the minimal
harboring a major histocompatibility complex (MHC) class I structural element of PG required for activation of the intracel-
specific epitope [12]. Either the structurally defined TLR2- lular protein NOD2 is N-acetylmuramyl-L-alanine-D-isogluta-
ligand Pam3CSK4 [13,14], TLR7-ligand 7-hydroxy-8-oxoade- mine (MDP, Figure 1, 1) [27-30]. Based on the effectiveness of
nine [15] or TLR9-ligand CpG DNA [13] were covalently the above mentioned TLR2/9-ligand conjugates, we hypothe-
bound to a model antigen, an ovalbumin derived peptide sized that constructs consisting of a NOD2 ligand covalently
comprising the MHC I epitope SIINFEKL, embedded in a bound to the ovalbumin derived model peptide (DEVA5K)
longer peptide motif (DEVSGLEQLESIINFEKLAAAAAK, could lead to similar enhanced immunological activity. Impor-
DEVA5K) [13,16]. We revealed that conjugates, in which tant issues in the design of such conjugates are the selection of a
Pam3CSK4 or CpG DNA were incorporated, showed an suitable MDP derivative and the position of the covalent
increased uptake of conjugated peptide. Increased DC matura- linkage between the NOD2-ligand and the peptide epitope [31-
tion and enhanced antigen presentation was achieved in com- 33]. Besides, the nature of the interaction between MDP and
parison to the mixture of the single peptide and ligands [13,14]. NOD2 receptor at the molecular level has not been established
yet. Therefore we considered different linkages between MDP
In the late 1990s the cytosolic nucleotide-binding oligomeriza- and the antigenic peptide (Figure 1) and we decided to hook up
tion domain (NOD) receptors [17] NOD1 [18] and NOD2 [19] the NOD2 ligand to either the N- or C-terminus. As a point of
were discovered to be intracellular PRRs [20,21]. NOD1 and attachment on the ligand we opted for a linker connected to the
NOD2 recognize specific parts of peptidoglycan (PG), found in anomeric center of the MDP or the carboxylic acid function of
the bacterial cell wall [22,23]. PG consists of a polysaccharide isoglutamine as viable conjugation sites. It is known that the
chain of β-(1–4) linked N-acetylglucosamine (GlcNAc) and size and orientation of the aglycon installed at the anomeric
Figure 1: MDP-antigen conjugates 2–5. DEVA5K = DEVSGLEQLESIINFEKLAAAAAK.
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center of MDP influences its immunological activity [18,34] the low solubility of MurNAc 10 and dipeptide 13, building
and therefore we selected an 3-azidopropanol spacer as conju- block 14 was obtained in 70% yield. Isolation and purification
gation handle with minimal steric bulk. With respect to the of key intermediate 14 was substantially facilitated by the
second conjugation site, i.e., the isoglutamine residue, it has finding that the compound could be efficiently precipitated from
been reported that condensation of this acid with an unnatural a MeOH/DCM/diethyl ether solvent mixture. The synthesis of
amine does not affect the immunological properties of MDP building block 16 started with the reduction of the azide in com-
[34]. A conjugate of the anticancer drug Paclitaxel with MDP pound 14 with PMe3 and subsequently the primary amine and
has been described using this conjugation locus, which not only Fmoc protected glutamic acid allyl ester were condensed under
showed antitumor activity but also immunostimulatory effects influence of HATU and DiPEA to give the orthogonally
[35]. Also, the commercially available immunomodulator protected compound 15 in 57% yield. To make 15 suitable for
Murabutide, a glutamine n-butyl ester derivative of MDP, SPPS, the allyl protective group was removed with Bu3SnH and
supports the notion that modifications on the isoglutamine are Pd(PPh3)4 under acidic conditions yielding compound 16 in
allowed [36]. Thus, MDP-antigen conjugates 2–5 were selected 72% [42].
as target molecules (Figure 1). In conjugate 2 the carboxylic
acid function of the isoglutamine of the MDP is linked to the For the immunological evaluation of conjugates 2–5 relevant
N-terminal amine of the antigenic peptide. In conjugate 3 the reference compounds are needed, and to this end we assembled
same acid function of the MDP connects to the C-terminal MDP derivative 20. On paper, acidic removal of the Boc and
lysine of the antigenic peptide. The 3-azidopropanol spacers at benzylidene protecting groups in 14 could lead to reference
the anomeric center of MurNAc in conjugate 2 and 3 remain compound 20 in a single step. However, treatment of 14 with a
unmodified. In conjugates 4 and 5 on the other hand the solution of 20% TFA in DCM was accompanied by hydrolysis
3-azidopropanol is functionalized with glutamic acid allowing of the glycosidic linkage. To suppress acid-mediated hydrolysis
conjugation to the antigenic peptide at the N-terminus for a stepwise deprotection procedure was followed in which the
conjugate 4 and at the C-terminus for conjugate 5.
benzylidene group was first replaced by electron-withdrawing
acetyl groups. This protective group pattern provides higher
We here describe the assembly of MDP building blocks, suit- acid stability of the glycosidic linkage and permits removal of
able for automated solid phase peptide synthesis (SPPS), their the Boc group using more stringent conditions. Thus, treatment
use in the assembly of the four MDP-antigen conjugates 2–5 of 14 with 60% aqueous acetic acid in the presence of two
and the immunological evaluation of the constructs.
equivalents of neopentyl glycol at 60 °C and careful monitoring
of the reaction progress led to compound 17 in 88% yield.
Acetylation of 17 then afforded compound 18. Stabilization of
the glycosidic linkage by the installment of electron-with-
Results and Discussion
Synthesis of the conjugates
The MDP-antigen conjugates 2–5 were prepared using an auto- drawing groups proved to be successful, and the treatment of 18
mated solid-phase peptide synthesis (SPPS) protocol. In all with 20% TFA in DCM resulted in compound 19 in 82% yield.
these syntheses commercially available Tentagel S RAM resin During the reaction only a minimal amount of hydrolysis was
and amino acids were applied. The synthesis of the required observed. The synthesis was continued by the treatment of 19
MDP building blocks 10 and 16 is depicted in Scheme 1. The with ammonia in MeOH and ensuing purification of the crude
3-azidopropanol spacer was attached to the anomeric center of product using HW40 gel filtration to result in reference com-
the glucosamine moiety using oxazoline 6 [37-39]. Deacetyl- pound 20 in 87% yield.
ation and subsequent installation of the benzylidene protective
group then gave alcohol 9. Coupling of 9 with (S)-2-chloro- With MurNAc building block 10 and 16 in hand, the solid-
propanoic acid in the presence of sodium hydride resulted in the phase peptide synthesis of the MDP-antigen conjugates 2–5 was
formation of protected MurNAc 10 in 93% yield [39,40]. Fully undertaken (Scheme 2). Commercially available Fmoc
protected MDP 14 was obtained by condensation of acid 10 and protected amino acids equipped with standard acid labile
dipeptide 13. To this end, Fmoc-protected tert-butyl glutamic protective groups were used. The side chain of the C-terminal
acid 11 was reacted with di-tert-butyl dicarbonate and trans- lysine of the antigenic peptide was protected with the methyl
formed into 12 [41]. In a one-pot procedure compound 12 was trityl (Mtt) protective group, allowing the modification of both
deprotected with DBU and after quenching the reaction mix- the N- or C-terminal end at the final stage of the synthesis. In a
ture with HOBt, the free amine was condensed with Fmoc- standard elongation cycle using HCTU as a coupling reagent,
protected alanine to give compound 13 in 82% yield. The same acetic anhydride as capping reagent and piperidine to remove
procedure was applied to condense MurNAc 10 with dipeptide the Fmoc-group, immobilized peptide 21 was assembled using
13, using the more reactive coupling reagent HATU. Despite fully automated peptide synthesis (Scheme 2). To obtain conju-
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Beilstein J. Org. Chem. 2014, 10, 1445–1453.
Scheme 1: a) TMSOTf, DCM, 83%; b) cat. NaOMe, MeOH, quant.; c) CSA, PhCH(OMe)2, MeCN, DMF, 86%; d) (S)-2-chloropropionic acid, NaH,
1,4-dioxane, 93%; e) Boc2O, NH4HCO3, pyridine, 1,4-dioxane, 99%; f) 1) DBU, HOBt, DCM; 2) Fmoc-L-Ala-OH, EDC, DIPEA, DCM, 82%; g) 1) 13,
DBU, HOBt, DCM; 2) 12, HATU, DIPEA, DCM, 70%; h) 1) Me3P (solution in THF), DMF, THF, 80%; 2) Fmoc-Glu-(OH)-OAllyl, HATU, DIPEA, DMF,
57%; i) Pd(PPh3)4, Bu3SnH, AcOH, DMF, 72%; j) 60% AcOH, H2O, neopentylglycol, 88%; k) Ac2O, pyridine; l) 20% TFA, DCM, 82% (2 steps);
m) NH4OH, MeOH, 87%.
gate 2 with MDP on the N-terminus of the peptide, peptide 21 Purification by RP-HPLC resulted in the isolation of pure
was consecutively elongated with Fmoc-D-isoglutamine, Fmoc- conjugate 2 (2% overall yield).
L-alanine and MurNAc 10. The coupling of MurNAc 10
involved a double coupling protocol, using HATU instead of In the next target conjugate 3, MDP occupies the C-terminal
HCTU as condensing agent. The obtained fully protected and position of the antigenic peptide. To obtain 3, the N-terminus of
immobilized precursor was treated with a cocktail of 95% TFA, immobilized peptide 21 was protected with a Boc-group by
2.5% TIS and 2.5% H2O to give, after precipitation in Et2O, treatment of 21 with 1 M Boc2O in NMP and two equivalents of
conjugate 2 and the hydrolyzed conjugate 22 in a 1:1 ratio. DIPEA (Scheme 2). Next, the resin was treated with a solution
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Scheme 2: a) 20% piperidine, NMP; b) Fmoc SPPS DEVA5K; c) Fmoc-D-isoGln-OH, HCTU, DIPEA, NMP; d) Fmoc-L-Ala-OH, HCTU, DIPEA, NMP;
e) 10, HATU, DIPEA, NMP; f) 95% TFA, 2.5% H2O, 2.5% TIS; g) Boc2O, NMP, DIPEA; h) 3% TFA, DCM. i) 16, HATU, DIPEA, NMP; j) 16, DIPEA,
HCTU, DMSO, DMF.
of 3% TFA in DCM to selectively remove the Mtt protective which turned out to be challenging. Changing the solvent mix-
group from the side chain of the C-terminal lysine. The ture from pure NMP to 20% DMSO in NMP led to 26 with a
resulting free amine was consecutively elongated with Fmoc-D- moderate coupling efficiency of 56% as judged from an Fmoc-
isoglutamine, Fmoc-L-alanine and MurNAc 10 as described for cleavage test of an aliquot of resin. The Fmoc protective group
conjugate 2. Subsequently, the resin was subjected to the in 26 was removed and the resin was elongated by automated
cleavage cocktail (95% TFA, 2.5% TIS and 2.5% H2O) to give SPPS with the DEVA5K motif resulting in the immobilized
conjugate 3 and the hydrolyzed conjugate 25 in a 1:1 ratio. fully protected conjugate. The same deprotection and cleavage
Purification by RP-HPLC gave pure conjugate 3 (2% overall conditions as described for 4 were used to isolate conjugate 5.
yield).
Treatment of the resin with the cleavage cocktail for a shorter
reaction time reduced the anomeric hydrolysis of the conjugate.
Conjugate 4 was obtained by the application of advanced Purification by RP-HPLC gave pure conjugate 5 in 6% overall
building block 16 (Scheme 1). En route to 4, peptide 21 was yield.
elongated with 16 in a coupling cycle using HATU as a
Immunological evaluation of the MDP-antigen
coupling reagent (Scheme 2). The solid-phase synthesis scheme
was concluded with the removal of the Fmoc protective group conjugates
resulting in the partially protected immobilized precursor of 4. The immunostimulatory activity of conjugates 2–5 was assessed
Acidic cleavage of all protecting groups and release of the in three different assays. In these assays, besides reference com-
target conjugate from the resin gave, after precipitation of the pound 20, also the unconjugated peptide, DEVA5K (29), and
crude product from Et2O at –20 °C and RP-HPLC purification, the Pam3Cys-antigen conjugate 30 were used as relevant
pure conjugate 4 in 2% overall yield.
control compounds [13]. In the first assay the NOD2 stimula-
tory potential of the conjugates was assessed. In the second and
To synthesize the C-terminal functionalized conjugate 5, Tent- third assay the conjugates were evaluated for their potential to
agel S RAM resin was first condensed with building block 16, induce maturation of dendritic cells (DCs) and their ability to
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support uptake followed by antigen processing and presentation affect the NOD2 stimulating activity significantly, although the
by DCs. The MHC class I molecule Kb on DCs derived from N-terminal conjugate appears slightly more active than the
C57BL/6 mice is able to present the SIINFEKL epitope C-terminal conjugated MDP.
(OVA257-264), which is embedded in the antigenic long
peptides. Presentation of the SIINFEKL-antigen can be The ability of conjugates 2–5 to induce DC maturation was
measured by adding a CD8+ T cell hybridoma (B3Z), evaluated by measuring interleukin 12 (IL-12p40) production
which is specific for the SIINFEKL epitope. This hybridoma by the cultured murine DC line, D1 [45]. IL-12 secretion by
contains a lacZ reporter construct linked to the nuclear factor of DCs is a good indicator for the immunostimulatory potential of
activated T cells (NFAT), enabling colorimetric measurement a compound, because this cytokine is essential for T cell stimu-
of the level of T cell receptor mediated activation of this T cell lation and differentiation upon presentation by these antigen
clone.
presenting cells. In Figure 3 it is shown that conjugates 2–5
produce amounts of IL-12 that are close to background level. In
The NOD2 activating capacity of conjugates 2–5 was tested agreement with the results of the NOD2 transfected HEK cells
using a stable NOD2 transfected human embryonic kidney conjugates 4 and especially 5 are more active than 2 and 3 indi-
(HEK293) cell line, in which the level of interleukin 8 (IL-8) cating that the MDP ligation locus affects the activity of the
production reflects the NOD2 stimulatory capacity of the com- constructs. Notably, reference Pam3Cys-conjugate 30 is much
pounds (Figure 2). MDP derivative 20 showed a substantial more potent than MDP-conjugates 4 and 5. A possible explan-
amount of IL-8 production, confirming that the β-azido- ation for the lack of strong maturating activity of the conju-
propanol modification on the anomeric position is allowed gates 4 and 5 is that these conjugates are not effectively inter-
[43,44]. Of conjugates 2–5 the conjugates 2 and 3 showed nalized and therefore only a very low concentration reaches the
activity close to background levels. In contrast, conjugates 4 cytosol resulting in a poor stimulation of the NOD2 receptor
and 5 induced IL-8 production at a level comparable to MDP [46].
derivative 20. The difference in the activity of conjugates 2 and
3 compared to 4 and 5 indicates that the attachment point of the Finally, the influence of the MDP-modification on the MHC
MDP to the antigenic peptide is important. Conjugation at the I-mediated presentation of the antigenic peptide was investi-
GlcNAc anomeric center of MDP as in conjugates 4 and 5 is gated using the SIINFEKL-specific T-cell hybridoma (B3Z)
more favorable than conjugation to the side chain of the L-iso- assay. The DCs were treated with constructs 2–5 and reference
glutamic acid residue (2, 3). The negative controls, peptide 29 compounds 29 and 30 (Figure 4). All constructs gave rise to
and conjugate 30, showed no activity indicating that the activi- antigen presentation, which indicates that the presentation is not
ties of 4 and 5 are a consequence of the presence of the MDP in affected by the condensation of an MDP to either C- or
these conjugates. Notably, the location of the ligand on either N-terminus of the antigenic peptide. Known conjugate 30
the C- or N-terminus of the peptide (4 vs 5) does not seem to containing TLR-2 ligand Pam3CSK4 showed, as previously
Figure 2: Potency of the NOD2-L antigen conjugates 2–5 in NOD2 transfected HEK cells.
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Figure 3: DC activation of the conjugates 2–5 and reference compounds 29 and 30.
Figure 4: Antigen presentation of the conjugates 2–5 and reference compounds 29 and 30.
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reported, an increased level of antigen presentation in compari- Conjugates of these lipophilic MDPs can therefore be attractive
son to peptide 29 [13].
vaccine modalities and efforts are currently underway in our
laboratory to investigate these.
Collectively, the data obtained from the assays described indi-
cate that the MDP-based conjugates 2–5 are poor maturating
agents for murine dendritic cells while antigen presentation
from the peptide part of the constructs is retained. Since the
NOD2 activity of constructs 4 and 5 is intact as determined by
the assay using the NOD-2 transfected cell line, the overall
failure of the described construct to mature DCs is probably not
caused by the lack of receptor binding. The NOD2 receptor is
located in the cytosol and the mechanisms underlying NOD2
ligand internalization and processing, before reaching the
receptor in the cytosol are not yet fully understood [46]. There
are indications that PG fragments are internalized via endocy-
tosis and subsequently processed and transported to the cytosol.
Because the antigen presentation assay indicates that the NOD2
modified peptides are processed correctly and presented by the
MHC class I molecules, we assume that the poor activity of the
MDP-conjugates is not (solely) due to the inability of these to
reach the cytosol of the DC. Presumably the NOD2 triggering
by MDP in the used murine DC cell line is comparatively inef-
fective as compared to the NOD2 transfected HEK cells.
Supporting Information
Supporting Information File 1
Full experimental details and characterization of all new
compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-148-S1.pdf]
Supporting Information File 2
NMR spectra.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-148-S2.pdf]
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