Synthesis of 2-alkoxy-8-hydroxyadenylpeptides: Towards synthetic epitope-based vaccines

Article abstract of DOI:10.1016/j.bmcl.2006.03.034

The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase synthesis combined with 'on-resin' Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evaluated in IL-12 production and antigen presentation assays.

Full text of DOI:10.1016/j.bmcl.2006.03.034

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3258–3261
Synthesis of 2-alkoxy-8-hydroxyadenylpeptides: Towards
synthetic epitope-based vaccines
a
b
a
b
Jimmy J. Weterings, Selina Khan, Gerbrand J. van der Heden, Jan W. Drijfhout,
b
a
c
b
Cornelis J. M. Melief, Herman S. Overkleeft, Sjoerd H. van der Burg, Ferry Ossendorp,
a,
a,
*
*
Gijsbert A. van der Marel and Dmitri V. Filippov
a
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, PO Box 9600,
300 RC Leiden, The Netherlands
b
2
c
Department of Clinical Oncology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
Received 31 January 2006; revised 13 March 2006; accepted 13 March 2006
Available online 11April 2006
Abstract—The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase
synthesis combined with ‘on-resin’ Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evalu-
ated in IL-12 production and antigen presentation assays.
Ó 2006 Elsevier Ltd. All rights reserved.
1
0a
Toll-like receptors (TLRs) are key targets in the devel-
1
lipopeptides (peptides containing a palmitoylated cys-
10b
teine residue) and nucleopeptides (hybrids of peptides
opment of well-defined, potent and selective vaccines.
TLRs recognize specific molecules associated with com-
mon pathogens, such as bacteria and viruses, and elicit
and CpG phosphorothioate DNA) have been reported
as structurally defined constructs designed for TLR-2
and TLR-9 recognition, respectively.
2
an inflammatory response. A range of TLRs (TLR1
through TLR11) have been discovered, however the ex-
act molecular identity of only a limited number of their
In this paper, we present the synthesis and biological
evaluation of hydroxyadenylpeptide hybrid structures
3, 4 and 5 (Fig. 1) containing the well-known major his-
tocompatibility complex (MHC) class I epitope from
ovalbumin (SIINFEKL, Figure 1, sequence in bold).
3
ligands has been established. The most notable
structurally defined ligands are lipopeptides having
a terminal N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-
propyl]cysteine (Pam Cys-OH) moiety, a ligand of
3
4
TLR2 , and oligodeoxynucleotides containing the CpG
5
motif, a ligand of TLR9. Interestingly, TLR7, that is
expressed intracellularly in the endosomes, can bind
The potent interferon-alpha inducer 2-alkoxy-8-hydroxy
1
1,12
adenine derivative 1 (Fig. 1)
is thought to act as a
6
11
small drug-like compounds such as imidazoquinolines
7
and guanine derivatives.
TLR7 ligand (TLR7-L). Structure–activity studies jus-
tify alteration of the 2-substituent in 1 such as in alky-
lazide derivative 2 which is suitable for chemoselective
attachment to alkyne functionalized peptides through
It has been previously discovered that covalent attach-
ment of Toll-like receptor ligands (TLR-L) to antigenic
proteins enables the development of model vaccines with
enhanced immunogenicity in comparison to a mixture
1
3,14
copper catalyzed Huisgen cycloaddition.
This
chemoselective transformation does not necessitate any
protective group manipulation on the exocyclic amino
group of hydroxyadenine and enables a straightforward
synthetic entry to the target peptide derivatives 3, 4 and
8
,9
of the two components. Up to now, however, only
5 (Fig. 1) with the TLR7-L attached to either the C- or
the N-terminus of the peptide.
Keywords: TLR-7; Peptide synthesis; Huisgen cycloaddition; Dendritic
cells; Antigen presentation.
*
Corresponding authors. Tel.: +31 71 5274353; fax: +31 71 5274307
D.V.F); e-mail: filippov@chem.leidenuniv.nl
The primary structure of the selected model peptides
1
reflects earlier findings indicating that for an optimal
5
(
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.034

J. J. Weterings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3258–3261
3259
NH2
N
H
N
O
O
NH2
Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-OH
O
N
H
N
N
O
R
N
O
N
Bn
3
O
N
N
N
N
O
N
O
1
2
: R = Me
Bn
: R = CH CH OCH CH N
2
2
2
2
3
O
NH2
N
NH2
Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Ala-Ala-Ala-Ala-Ala-Lys-NH2
H
N
H
N
N
N
N
N
N
O
O
N
O
4
O
N
O
O
N
N
N
O
O
O
Bn
Bn
5
Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Ala-Ala-Ala-Ala-Ala-Lys-NH2
Figure 1. 2-Alkoxy-8-hydroxyadenine derivatives and peptide conjugates thereof.
NH2
N
Cl
N
immune response a CTL (cytotoxic T lymphocyte)-
epitope (SIINFEKL in this study) has to be embedded
in a longer peptide motif. This results in predominant
presentation of the epitope (after proteolytic processing)
on professional antigen-presenting cells (such as dendrit-
ic cells) and allows for exposure to the immune system in
the context of MHC class I molecules. An important
feature in the processing of polypeptides towards
MHC class I epitopes is the involvement of the protea-
some, which is responsible for the generation of the
C-terminus of the epitope (here L in SIINFEKL). A
peptide epitope targeted for presentation in the MHC
class I route should not interfere with this process. In
practice, this entails that either the conjugate terminates
in the correct C-terminal residue or has a C-terminal
peptide extension motif. Conjugate 3 meets the first cri-
terion (it features the SIINFEKL motif at the C-termi-
nus) thereby allowing the introduction of the adenine
b
N
N
N
N
N
Cl
N
R
Cl
8
6: R = H
a
7: R = Bn
N3
NH2
N
O
N
R1
c
O
R
N
N
OH
9
10:R = (OCH2CH2)3N3;
d
1
R = H
N3
O
1
1:R = (OCH CH ) N ;
NH2
2
2 3
3
H
1
R = Br
O
N
N
O
O
N
N
e
2
moiety to the N-terminus. Compounds 4 and 5 contain-
ing the penta-alanine elongated peptide, suitable for
proteasomal processing, are modified with the adenine
moiety at the N- and C-termini, respectively.
Scheme 1. Synthesis of the key building block 2. Reagents and
conditions: (a) BnBr, TBAF, 2 h, 61%; (b) NH
/MeOH, 120 °C, 15 h,
7%; (c) 9, DME, reflux, NaH, 69%; (d) Br /DCM, 2 h, 78%; (e) 3 N
HCl, reflux, 3 days.
3
9
2
The assembly of conjugates 3, 4 and 5 (Fig. 1) requires
the availability of azide derivative 2 (Scheme 1). The
synthesis of 2 is based on procedures published for the
The synthesis of 3 (Scheme 2) started with a stepwise
assembly of the peptide chain on the commercially
available TFA sensitive Tentagel S PHB resin preloaded
with Fmoc-Leu-OH (12). Repetition of the coupling
1
1,12
preparation of related 9-alkyl-2-alkoxyadenines
and commences with N-alkylation of 2,6-dichloropurine
a
cycle, entailing cleavage of the N -Fmoc-groups with
1
6
(
6) with benzyl bromide under the agency of TBAF to
piperidine and introduction of the amino acids, as well
as the electron-deficient propiolic acid residue, using
HCTU (2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetra-
give 9-benzyl-2,6-dichloropurine 7 together with the 7-
benzylated isomer. Chromatographic separation led to
the isolation of both isomers in 61% (7) and 36% yield,
19
methylaminium hexafluorophospate) assisted activa-
tion led to immobilized 13. Subsequent attachment of
the hydroxyadenine moiety was executed by treatment
1
7
respectively (Scheme 1). Regioselective ammonolysis
by heating of 7 with methanolic ammonia in a sealed
tube under microwave irradiation for 15 h at 120 °C
yielded 6-amino-2-chloropurine 8 in 97% yield. Treat-
1
8
ment of 8 with 2-[2-(2-azidoethoxy)ethoxy]ethanol
9) and NaH afforded the corresponding 2-alkoxyade-
(
O
a,b
O
*
O
O
nine 10. Selective bromination at the C(8)-position was
carried out with bromine to provide 11 in 78% yield.
AA
Fmoc N
H
HN
Tentagel PHB
18
O
1
2
13
*
AA 18
:
Asp(OtBu)-Glu(OtBu)-Val-Ser(tBu)-
Gly-Leu-Glu(OtBu)-Gln(Trt)-Leu-
Glu(OtBu)-Ser(tBu)-Ile-Ile-Asn(Trt)-
Phe-Glu(OtBu)-Lys(Boc)-Leu
c-e
Treatment of 11 with 3 N HCl at reflux gave 9-benzyl-
8
2
-hydroxy-2-(2-[2-(2-azidoethoxy)ethoxy]ethoxy) adenine
. Compound 2 was found to be of satisfactory purity
62%, RP-HPLC) and contained as detectable side prod-
3
(
Scheme 2. Solid-phase synthesis of compound 3. Reagents and
conditions: (a) SPPS; (b) propiolic acid, HCTU, DiPEA; (c) 2, CuI,
ucts only compounds lacking the azidotriethyleneglycol
tail, and we therefore opted to use the mixture in the
solid-phase Huisgen reaction without purification.
DiPEA, NMP; (d) TFA/TIS(triisopropylsilane)/H O (95:2.5:2.5); (e)
2
RP-HPLC.

3260
J. J. Weterings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3258–3261
(
48 h) of resin 13 suspended in NMP with alkylazide
functionalized building block 2 in the presence of CuI
and DiPEA. Target compound 3 was released from
the solid support and simultaneously deprotected by
2
0
treatment with TFA and isolated by ether precipita-
tion. Crude hydroxyadenylated peptide 3 was analyzed
by LC–MS using a C -column. No trace of the propiol-
1
8
yl peptide was detected, suggesting that the cycloaddi-
tion reaction went to completion. Purification using a
semi-preparative RP-HPLC column gave homogeneous
2
1
3
in 4.3% overall yield.
After the successful completion of the synthesis of con-
jugate 3, very similar methodology was adopted for the
preparation of the structurally different conjugates 4 and
Figure 2. Potency of 8-hydroxyadenine derivatives in dendritic cells,
activation determined by the level of induced IL-12p40.
5
2
(Fig. 1). Copper(I) catalyzed cycloaddition of azide
to alkyne functionalized peptide resin 15 (Scheme 3),
obtained via SPPS initiated on immobilized Fmoc-
Lys(Mtt) residue (14, Scheme 3), gave after TFA treat-
ment and purification conjugate 4 in an overall yield
2
1
of 11.3%. Alternatively, Fmoc-Lys(Mtt) resin 14 was
converted into immobilized 16 by the following proce-
dure: HCTU/DiPEA assisted Fmoc-SPPS, Boc O/Di-
2
PEA treatment to block the N-terminus, 1% TFA/
2
2
DCM treatment to selectively cleave the Mtt group
from the side chain of the C-terminal lysine residue
and finally HCTU/DiPEA mediated introduction of
the propiolic acid residue. Subsequent CuI catalyzed
Huisgen cycloaddition followed by TFA treatment and
RP-HPLC purification furnished 5 in 2.9% overall
2
1
yield.
We next investigated the ability of the pure synthetic
8
3
(
-hydroxyadenine derivative 2 and the modified peptides
, 4 and 5 to induce production of the T helper cell
Th1)-activating cytokine, interleukin-12 (IL-12p40).
Figure 3. Comparing MHC class I antigen presentation of conjugated
and non-conjugated peptides. Dendritic cells were incubated for 2 h in
the presence of either conjugate 3 or a mixture of compound 2 with 18
meric parent peptide containing the cytotoxic T-cell epitope SIIN-
FEKL. Subsequently cells were washed and incubated with B3Z T-cell
hybridoma for 24 h before quantification of the T-cell response.
1
1
The known derivative 1 and the established TLR7 li-
6
gand R848 were included in the assays as reference
compounds. Bone-marrow derived dendritic cells
(
DCs) were incubated with the indicated substances
and IL-12p40 production in the culture supernatant
was measured after either 24 or 48 h. As evident from
Figure 2 both 1 and 2 efficiently induce IL-12p40
production. Conjugation of the peptides to the TLR7 li-
gand (compounds 3, 4 and 5) led to complete abolishment
of the IL-12p40 production. Next, we investigated the ef-
fect of the hydroxyadenine modification on the antigen
presentation. As evident from Figure 3, specific T cell re-
sponse against the SIINFEKL epitope can be elicited
when DCs are treated with either TLR7-L-conjugate 3
or the free parent peptide in a mixture with compound
2. A pronounced enhancement in antigen presentation
was observed with the conjugate peptide compared to
the mixture. Exactly the same effect was observed when
conjugates 4 and 5 were compared to the parent peptide
Tentagel
Mtt
RAM
NHMtt
a,b
O
*
O
HN AA
NH
23
N LysNH
Fmoc N
15
H
H
O
1
4
e,f,g
O
a,c,d,b
NH
4
e,f,g
*
O
H
N
BocHN AA 2
5
3
N
23
mixed with 2.
H
O
1
6
*
AA 2
:
3
Asp(OtBu)-Glu(OtBu)-Val-Ser(tBu)-Gly-Leu-Glu(OtBu)-
Gln(Trt)-Leu-Glu(OtBu)-Ser(tBu)-Ile-Ile-Asn(Trt)-Phe-
Glu(OtBu)-Lys(Boc)-Leu-Ala-Ala-Ala-Ala-Ala
In summary, an efficient solid-phase synthesis of three
different 2-alkoxy-8-hydroxy adenyl peptide conjugates
(
3, 4 and 5) is presented. In comparison with a mixture
Scheme 3. Solid-phase synthesis of compounds 4 and 5. Reagents and
conditions: (a) SPPS; (b) propiolic acid, HCTU, DiPEA; (c) Boc O,
DiPEA; (d) 1% TFA/DCM; (e) 2, CuI, DiPEA, NMP; (f) TFA/TIS/
O (95:2.5:2.5); (g) RP-HPLC.
of their individual components, these conjugates give
rise to enhanced antigen presentation in vitro but lack
the ability to induce DC activation. As the in vitro
2
H
2

J. J. Weterings et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3258–3261
3261
antigen presentation assay is independent of the matura-
tion status of the antigen-presenting cells, the enhance-
ment of the antigen presentation of the conjugates
may be explained by the improved targeting of the con-
jugate to the DCs compared to the free peptide. The lack
of DC activation by the conjugates is likely due to either
the poor binding of the conjugated ligand to TLR7 as a
result of steric hindrance of the peptide moiety, the im-
paired intracellular trafficking of the conjugated ligand
compared to free 2, or both.
7. Lee, J.; Chuang, T. H.; Redecke, V.; She, L. P.; Pitha, P.
M.; Carson, D. A.; Raz, E.; Cottam, H. B. Proc. Natl.
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. Tighe, H.; Takabayashi, K.; Schwartz, D.; Marsden, R.;
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939.
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. Wille-Reece, U.; Flynn, B. J.; Lore, K.; Koup, R. A.;
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0. (a) Spohn, R.; Buwitt-Beckmann, U.; Brock, R.; Jung, G.;
Ulmer, A. J.; Wiesmuller, K. H. Vaccine 2004, 22, 2494;
According to this line of reasoning the stimulatory
capacity of the conjugates can be restored by the intro-
duction of cleavable linker between the TLR-ligand and
the peptide moiety allowing release of the ligand after
internalization. The internalization itself can be im-
proved by inclusion of functionalities known to enhance
endosomal uptake.
(
b) Daftarian, P.; Sharan, R.; Haq, W.; Ali, S.; Longmate,
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Ogita, H.; Takaku, H.; Sajiki, H.; Hirota, K.; Kawakami,
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Sajiki, H.; Isobe, Y.; Takaku, H.; Tobe, M.; Ogita, H.;
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Chem. 2002, 45, 5419.
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K. B. Angew. Chem. Int. Ed. 2002, 41, 2596.
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H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8,
1
1
The design, synthesis and evaluation of such next-gener-
ation conjugates are now in progress and will be reported
in due course.
1
Acknowledgment
1
128.
This work has been funded by Netherlands Organisa-
tion for Scientific Research (NWO) as a part of the
1
5. Zwaveling, S.; Mota, S. C. F.; Nouta, J.; Johnson, M.;
Lipford, G. B.; Offringa, R.; van der Burg, S. H.; Melief,
C. J. M. J. Immunol. 2002, 169, 350.
‘
From Molecule to Cell’ program.
1
1
1
6. Brik, A.; Wu, C. Y.; Best, M. D.; Wong, C. H. Bioorg.
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Supplementary data
5
2, 5625.
8. Li, J.; Zacharek, S.; Chen, X.; Wang, J. Q.; Zhang, W.;
Detailed experimental procedures and characterization
for all new compounds. This material is available free
of charge via the Internet at http://www.elsevier.com.
Supplementary data associated with this article can
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1. Conjugates 3, 4 and 5 were dissolved in 30% hexafluoro-
isopropanol/water and analyzed with LC/MS. Gradients
of B in A/C (9:1) were applied over 15 min. Solvent
system: A, 100% water; B, 100% acetonitrile; C, 1% TFA
in water.
2
006.03.034.
References and notes
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. Pashine, A.; Valiante, N. M.; Ulmer, J. B. Nat. Med. 2005,
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1
Compound 3: LC/MS 10–70% B, t = 13.44 min. ESI-MS:
R
+
[M+H] : 2530.6 (calcd 2530.5).
2
2
Compound 4: LC/MS 10–70% B, t = 12.47 min; ESI-MS:
R
2
[M+H] : 1506.8 (calcd 1506.5).
+
3
4
Compound 5: LC/MS: 10–70% B, t = 12.35 min; ESI-
R
2
MS: [M+H] : 1506.8 (calcd 1506.5).
+
5
6
. Krieg, A. M. Nat. Med. 2003, 9, 831–835.
22. Bourel, L.; Carion, O.; Gras-Masse, H.; Melnyk, O. J.
Pept. Sci. 2000, 6, 264.
23. The antigen presentation data for compounds 4 and 5 are
included in Supplementary data (Figure S6).
. Hemmi, H.; Kaisho, T.; Takeuchi, O.; Sato, S.; Sanjo, H.;
Hoshino, K.; Horiuchi, T.; Tomizawa, H.; Takeda, K.;
Akira, S. Nat. Immunol. 2002, 3, 196.