Immobilization by Surface Conjugation of Cyclic Peptides for Effective Mimicry of the HCV-Envelope E2 Protein as a Strategy toward Synthetic Vaccines

Article abstract of DOI:10.1021/acs.bioconjchem.7b00755

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e., epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate surfaces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To the best of our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity toward their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key binding residues by simply changing the amino acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key binding residue for binding monoclonal antibody V3.2.

Full text of DOI:10.1021/acs.bioconjchem.7b00755

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Article
Immobilization By Surface Conjugation Of Cyclic Peptides For Ef-fective Mimicry Of The HCV-Envelope E2 Protein As A Strategy Towards Synthetic Vaccines
Tom Meuleman, James Dunlop, Anna Owsianka, Helmus Van De Langemheen, Arvind H Patel, and Rob Liskamp
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00755 • Publication Date (Web): 31 Jan 2018
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Figure 3.
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Immobilization By Surface Conjugation Of Cyclic Peptides For Ef-
fective Mimicry Of The HCV-Envelope E2 Protein As A Strategy
Towards Synthetic Vaccines
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Theodorus (Tom) J. Meuleman [a], James I. Dunlop [b], Anna M. Owsianka [b], Helmus van de Langemꢀ
heen [a], Arvind H. Patel *[b], Rob M. J. Liskamp *[a]
[a]
T. J. Meuleman, Dr. H. van de Langemheen, Prof. R. M. J. Liskamp
School of Chemistry, University of Glasgow
Joseph Black Building, University Avenue,
Glasgow G12 8QQ (UK)
Eꢀmail: robert.liskamp@glasgow.ac.uk
[b]
Dr. J. I. Dunlop, Dr. A. M. Owsianka, Dr. A. H. Patel
MRCꢀUniversity of Glasgow Centre for Virus Research, Garscube Campus
Sir Michael Stoker Building, 464 Bearsden Road,
Glasgow G61 1QH (UK)
Eꢀmail: arvind.patel@glasgow.ac.uk
ABSTRACT: Mimicry of the binding interface of antibodyꢀantigen interactions using peptideꢀbased modulators (i.e. epitope mimꢀ
ics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward
fashion, thereby allowing for highꢀthroughput analysis. The design of epitope mimics is highly influenced by their spatial configuꢀ
ration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be
implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a
thiolꢀmoiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugatꢀ
ed via the free thiolꢀmoiety on maleimideꢀactivated plate surfaces. The resulting covalent, uniform, and oriented coated surface of
cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry
of an antigenꢀantibody interaction of the HCV E2 glycoprotein. To our knowledge, the benefit of cyclized peptides over linear pepꢀ
tides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated
specificity towards their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of
epitope mimics shows potential for highꢀthroughput screening of keyꢀbinding residues by simply changing the aminoꢀacid sequencꢀ
es within synthetic peptides. In this way, leucineꢀ438 has been identified as a keyꢀbinding residue for binding monoclonal antibody
V3.2.
INTRODUCTION
erating NAbs against this glycoprotein is not straightforward
as it contains highly variable immunogenic domains. Antibodꢀ
ies against these regions tend to be strainꢀspecific and unable
to prevent infection by a different strain of HCV. These variaꢀ
ble epitopes serve as decoys to distract the immune system. In
addition, HCV E2 contains 11 Nꢀlinked glycans that can shield
certain epitopes, thereby preventing binding of antibodies.7
Nevertheless, numerous NAbs have been isolated from patient
sera that are able to inhibit infection by a broad range of HCV
isolates. Most of these broadlyꢀneutralizing antibodies bind to
conserved, discontinuous regions within the HCV E2 glycoꢀ
protein and block its interaction with the CD81 receptor.8 Theꢀ
se regions are designated as broadly neutralizing epitopes.5, 9ꢀ11
The availability of structural information on these HCV E2
neutralizing epitopeꢀantibody interactions might be crucial for
future vaccine design.5, 10, 12, 13
Hepatitis C Virus (HCV) poses a global threat and is estiꢀ
mated to have infected over 3% of the world’s population.1 So
far its high mutation rate and intrinsic immune evasive strateꢀ
gies have hampered development of therapeutic and prophyꢀ
lactic vaccines. Despite this and the availability of novel antiꢀ
virals2 there is an urgent need for the development of HCV
vaccines,3 since drugs do not prevent reinfection4 and their
treatment is expensive.3 The potential of these possible future
vaccines is underlined by the existence of antibodies capable
of preventing viral infection (i.e. Neutralizing Antibodies
(NAbs)).5
Evidently, the HCV E2 envelope glycoprotein is of major
importance for development of HCV vaccines,6 as it is inꢀ
volved in viral attachment and entry, and is therefore an accesꢀ
sible target located on the surface of the virion. However, genꢀ
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Bioconjugate Chemistry
A decade ago we introduced a chemical biology perspective
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for designing synthetic vaccines by describing a strategy for
mimicry of discontinuous protective epitopes using a synthetic
scaffold for obtaining a 'PEPTAC' molecular construct, which
acted as a synthetic vaccine and protected mice against infecꢀ
tion with B. Pertussis.14 Although this was a very promising
result, we think in order to further improve chances for ultiꢀ
mately a successful synthetic HCV vaccine, we have to mimic
neutralizing epitopes present in E2 as closely as possible and
ligate them on a diversity of suitable scaffold molecules.15ꢀ17
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The required scaffold molecule should mimic the structural
integrity that is normally provided for by the majority of a
protein (Figure 1). Thus, the resulting epitope containing proꢀ
tein mimic will only present the essential epitope(s) and will
not contain any shielding or immunogenic decoy domains.
Consequently these protein mimics have the potential to lead
to synthetic and more efficient vaccines.18,19
Maleimide activated microplate
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Figure 2. Chemoselective approach for covalent immobiliꢀ
zation of epitope mimics in a uniform and specifically oriented
fashion via thiol addition to maleimide.
In this research we show the importance of cyclization of
peptides as a conformational constraint for effective mimicry
of neutralizing epitopes from HCV E2. To this end, more flexꢀ
ible, linear peptides were compared with their constrained,
cyclic counterparts. These peptide constructs were obtained
via benzylic bromide derivatives equipped with a tetraethylene
glycol (TEG) spacer containing a tritylꢀprotected thiolꢀmoiety
(7 and 8, respectively). After liberation of the thiol moiety,
instead of random nonꢀcovalent adsorption, a covalent coating
of epitope mimics on polystyrene plates was obtained using
commercially available Pierce® maleimide activated 96ꢀwell
plates (Figure 2).
Figure 1. Epitope mimicry, in which essential peptide loops
of a discontinuous epitope are ligated on a molecular scaffold.
Conformational constraints are probably very important in
the design of epitope mimics, since they can induce or enforce
conformations of epitopes as they are present in the native
protein.15 Many epitopes are comprised of loopꢀlike structures
and mimicry of loopꢀlike peptide sequences is best achieved
by cyclization. Furthermore, introduction of conformational
constraints by cyclization has been found to improve rigidity,
stability, bioavailability, and biological activity of peptideꢀ
based therapeutics.14, 17 Therefore, constrained peptides are to
be preferred in the design of adequate and effective epitope
mimics.20ꢀ25 However, to our knowledge, this favorable effect
on epitope mimicry by the introduction of conformational
constraints by cyclization has not been rigorously investigated
so far.
The availability of a human/mouse antibody variant of the
broadly neutralizing human monoclonal antibody (MAb)
HC84.1,9 as well as weakly neutralizing mouse antibody V3.2
(to be published), enabled us to evaluate our epitope mimics
using ELISA and the impact of peptide cyclization for effecꢀ
tive mimicry.
RESULTS AND DISCUSSION
The cornerstone of studying antigenꢀantibody interactions
and the identification of promising neutralizing epitopes is the
enzymeꢀlinked immunosorbent assay (ELISA). With respect
to this, novel isolated NAbs, obtained from immunization
studies or patients, are tested by ELISA to verify binding.9, 26,
Synthesis of the peptide cyclization and linear linker
with a maleimide reactive tetraethylene glycol (TEG) spac-
er. Recently, we introduced a bisbromobenzyl azide derivative
for cyclization of dicysteine containing peptides and subseꢀ
quent Cuꢀcatalyzed azideꢀalkyne cycloꢀaddition to alkyne conꢀ
taining scaffold molecules.14, 17 Here a related bisbromobenzyl
derivative was required, having instead of an azide functionꢀ
ality a TEG moiety for improved water solubility and a (proꢀ
tected) thiol group for reaction with maleimide moieties preꢀ
sent on the surface of an ELISA plate.31
27
Key binding residues of antigenꢀantibody interactions are
identified (i.e. epitope mapping) using mutagenesis studies, in
which ELISA is used to assess the altered binding affinity of
antibodies towards different mutants.9, 28 Conventional ELISA
involves nonꢀcovalent adsorption of antigen(s) on polystyrene
plates, which likely results in a heterogeneous surface of difꢀ
ferently adsorbed antigen molecules. Thus, these will probably
interact with different affinities with antibodies.29 In addition,
adsorption of antigens on polystyrene plates could potentially
perturb the structure of the antigen, thereby also influencing
antibody binding. Gori et al. have shown the importance of
accurate control of covalent immobilization and generating a
uniform and oriented surface for proper gauging of antigenꢀ
antibody interactions using peptide sequences.30
Thus, TEG 1 was monotosylated to tosylate 2 (61 %) folꢀ
lowed by substitution using crude tritylꢀthiol 3 leading to
tritylꢀTEGꢀsulfide 4 (quant.). Next, the cyclization linker moiꢀ
reaction with 1,3,5ꢀ
tris(bromomethyl)benzene 5 and trityl protected cyclization
linker 7 was obtained in a moderate yield (33%) (Scheme 1).
ety was introduced in
a
Similarly, tritylꢀTEGꢀsulfide
4 was reacted with 1,3ꢀ
bis(bromomethyl)benzene 6, to provide trityl protected linear
linker 8 also in a moderate yield (43%). Diꢀ and triꢀsubstituted
products were observed during the reaction of cyclic linker 7
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and linear linker 8, which at least partially explained the lower synthesized for the preparation of cyclic peptide construct 20
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yields of the mono substituted products. The availability of
both scaffolds supporting cyclized or linear peptide constructs
was essential for a rigorous evaluation of the constraining
effects by peptide cyclization.
to serve as a negative control, which should not bind to the
available MAbs HC84.1 and V3.2.
Synthesis of the epitope mimics for surface conjugation.
Reaction of the thus synthesized precursor peptides with either
linker construct 7 or 8 led to cyclic peptide constructs 15, 17,
19 and 20 and linear peptide constructs 16 and 18, respectively
(Scheme 2).
Scheme 1. Synthesis of PEGꢀbased thiol linkers for peptides
cyclization/attachment and conjugation to plates.
9
Scheme 2. Alkylation of precursor peptides with the linker
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constructs 7 or 8.
This alkylation procedure is convenient since it can be carꢀ
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tides exhibit good water solubility then normally NH4HCO3 in
a mixture water/acetonitrile can be used. However, since our
peptides were much more hydrophobic, DMF had to be inꢀ
cluded for dissolving the peptides and the alkylation approach
was optimized to dissolve the peptide and the linker in a conꢀ
centration of 1 mM in aqueous NH4HCO3 buffer (20 mM; pH
7.9) in dimethylformamide (DMF) (75 %). Immediately after
alkylation of the peptide on the linker, the trityl group was
removed to liberate the thiol functionality.
Alkylation of cysteine containing peptides with linkers 7
or 8. The required monoꢀ or diꢀ cysteine containing peptides
were obtained by solid phase peptide synthesis. Their amino
acid sequences originated from Epitope II present in HCV E2
glycoprotein (N434 TGWLAGLFYQHK446, residues in bold
are highly conserved across genotypes).9, 10, 32, 33 Epitope II is a
neutralizing immunogenic domain recognized by NAb
HC84.1 and weakly by NAb V3.2 (to be published). Epitopes
of MAb HC84.1 and V3.2 have been mapped to HCV E2 resiꢀ
dues 439 to 4469, 10 and residues 435 to 442 (to be published),
respectively. The available sequence information of the differꢀ
ent HCV genotypes/strains allowed for the optimization of
epitope II towards consensus sequences corresponding to resiꢀ
dues 436 to 448 and 430 to 444 of peptides shown in Table 1,
which cover the multiple genotypes of HCV.
Purification of the epitope mimics was achieved by preparaꢀ
tive reverse phase HPLC. The TEG spacer improved the aqueꢀ
ous solubility of the cyclic epitope mimics (15, 17, 19, and
20), slightly. The linear epitope mimics (16 and 18) showed a
significantly better solubility upon attachment of the TEG
containing linker. Nevertheless, all epitope mimics were obꢀ
tained in reasonable purity and yields, ranging between 90ꢀ
95% purity overall with 4ꢀ17% yield for the cyclic peptide
constructs 15, 17, 19, and 20 and 4ꢀ30% yield for the linear
peptide constructs 16 and 18. In our group, Van de Langemꢀ
heen et al. recently developed polar hinges that may be used
for future improvement of the general solubility of more hyꢀ
drophobic peptides in cyclization reactions.34
Peptides 9 (cp), 11 (cp), 13 (cp), and 14 (cp) served as cyꢀ
clization precursor (cp) peptides for the preparation of the
cyclic peptide constructs 15, 17, 19, and 20, respectively, and
were synthesized with a cysteine residue at both Nꢀ and Cꢀ
termini. Linear precursor (lp) peptides 10 (lp) and 12 (lp) conꢀ
taining one cysteine residue were synthesized to prepare the
linear peptide constructs 16 and 18. In addition, peptide 14
(cp) was designed with a scrambled aminoꢀacid sequence and
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Bioconjugate Chemistry
Table 1. Overview of the synthesized peptidic antigen sequences based on Epitope II of HCV E2 glycoprotein. cp: cyclization
precursor peptide, lp: linear precursor peptide. The cpꢀsequences have a Nꢀ and Cꢀterminal cysteine residue for cyclization whereas
the lp sequences have just one Cꢀterminal residue for attachment to the spacer. Peptide 14 has the scrambled sequence for preparaꢀ
tion of the negative control 20.
1
2
3
4
5
6
7
8
9
C G436 W
Ac–
V
V
L
A
A
A
A
A
K
G
G
G
G
G
Y
L
L
L
L
L
A
F
F
F
F
F
F
Y
Y
Y
Y
Y
W
Y
H
H
C
C
H
G
K
K
F
F
N448
C
C
–
NH2
9 (cp)
10 (lp)
11 (cp)
12 (lp)
13 (cp)
14 (cp)
G
G
G
436 W
Y
N448
Ac–
–
NH2
C N430 E
–
S
S
L
L
N
N
T
T
W
Y444
Y444
Y
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–
NH2
Ac
430E
W
L
Ac–
–
NH2
K
N
C G436 W
–
L
F
Y
N448
C
C
–
–
NH2
NH2
Ac
Ac
–
C H
F
G
L
N
L
Immobilization by surface conjugation to immobilized
maleimide residues. To improve conventional ELISA, the
individual epitope mimics were covalently ligated onto maꢀ
leimideꢀactivated plates. To verify antibody recognition of the
cyclic epitope mimics (15, 17, and 20) coated plate surface,
plates were incubated with a threeꢀfold dilution series of
MAbs HC84.1 and V3.2. It was observed that binding of MAb
HC84.1 was specific towards 15 (Fig. 3A) and binding of
MAb V3.2 was specific towards 17 (Fig. 3B). As expected, the
negative control containing a scrambled peptide sequence (20)
did not bind either MAb HC84.1 or V3.2. As mentioned beꢀ
fore, cyclic epitope mimics 15 and 17 are both based on the
epitope II region. However, the two MAbs HC84.1 and V3.2
bind epitope II via different residues. Therefore, using the
different synthetic peptides 9 (cp) or 11 (cp) it is possible to
demonstrate selectivity of the MAbs towards their respective
cyclic epitope mimics 15 and 17 (Fig. 3).
A
B
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
0.0
0.1
1.0
10.0
100.0
0.1
1.0
10.0
100.0
1000.0
Log[cV3.2 (ng/mL)]
Log[cHC84.1 (ng/mL)]
AcꢀC G436 W V A G L F Y Y H K F N448 CꢀNH2
AcꢀC N430 E S L N T G W L A G L F Y Y444 CꢀNH2
AcꢀC H F G K Y A F W L G N Y L CꢀNH2
15
20
17
Figure 3. ELISA comparing the immobilized cyclic epitope mimics 15 and 17 with the immobilized cyclic scrambled epitope 20
using MAbs HC84.1 and V3.2. (A): MAb HC84.1 (3ꢀfold dilution over 7 steps [111.1 – 0.2 ng/mL]); (B): MAb V3.2 (3ꢀfold diluꢀ
tion over 7 steps [37.0 – 0.1 ng/mL]). Background signal (no MAb) was subtracted. The data is represented as a percentage relative
to the highest absorbance being set to 100% per individual experiment (A: 15; B: 17). Data was collected from 3 to 5 independent
experiments, each performed in duplicate. Error bars represent the standard deviation.
Impact of peptide cyclization for adequate mimicry of
E2 peptide loops. To understand the impact of cyclization in
mimicry of epitopes, we studied the binding of MAbs
HC84.1 and V3.2 to cyclic and linear epitope mimics. To
this end, the cyclic (15 and 17) and linear (16 and 18)
epitopes mimics were ligated separately on maleimideꢀ
activated plates. The covalently ligated surfaces were incuꢀ
bated with a threeꢀfold dilution series of MAbs HC84.1 and
V3.2. A significant difference in antibody binding was obꢀ
served between the immobilized cyclic and linear epitope
mimics, for both MAbs HC84.1 and V3.2 (Fig. 4). MAb
HC84.1 successfully bound cyclic epitope mimic 15, but did
not show any binding to the corresponding linear epitope
mimic 16 (Fig. 4A). In addition, MAb V3.2 was capable of
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Bioconjugate Chemistry
Page 16 of 23
binding cyclic epitope mimic 17 but showed almost no bindꢀ partially an αꢀhelix in solution or an αꢀhelix was induced
1
2
3
4
5
6
7
8
ing to the corresponding linear epitope mimic 18 (Fig. 4B).
These results strongly underline the importance of peptide
cyclization for antibody recognition, which in turn advocates
peptide cyclization for accurate mimicry of epitopes. The
difference between MAb HC84.1 not binding linear epitope
mimic 16 at all and MAb V3.2 binding linear epitope mimic
18 to a minor extent suggests that the importance of peptide
cyclization could be sequence dependent. Perhaps, certain
linear peptides readily resemble the conformation of cyclized
peptides.
upon binding to HC84.1. In this work, the linear epitope
mimic 16 showed a major detrimental effect on binding
MAb HC84.1, which could indicate the inability of linear
precursor peptide 10 (lp) to form a necessary αꢀhelical
structure while alkylated on the linker. However, binding of
MAb HC84.1 was achieved by immobilized cyclic epitope
mimic 15, which suggests that the required interacting conꢀ
formation of the flexible linear peptide might be enforced by
cyclization. Since epitope mimicry is based on individual
peptide sequences of proteins being grafted on molecular
scaffolds, it is inevitable that the intrinsically adopted conꢀ
formations of linear peptides will be influenced. Therefore,
conformational constraints are necessary to preꢀorganize the
peptides into the required conformations. Nevertheless, it is
not assumed that our cyclized peptides behave similarly as
stapled peptides in which the αꢀhelix character is enhanced
by side chain cyclization, since stapling usually involves
cyclization by Ring Closing Metathesis (RCM) of (diꢀ
substituted) amino acid residues i and i+4 or residues i and
i+7. In peptide 15 side chains of residues i and i+14 are conꢀ
nected.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cyclization of our peptide sequence resulted in epitope
mimics that exhibit a higher affinity towards antibodies. This
serves as direct evidence that conformational constraints in
peptides resulted in improved mimicry. Nevertheless, the
flexibility of linear peptides might allow certain conforꢀ
mations to mold themselves onto binding interfaces, similar
to a lock and key model. Krey et al. have successfully coꢀ
crystalized human MAb HC84.1 bound to a linear peptide
based on the Epitope II region of HCV E2.10 The human
MAb HC84.1 has been shown to bind the peptide in a αꢀ
helical conformation. Perhaps, the linear peptide has adopted
A
B
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
0.1
1.0
10.0
100.0
1000.0
0.1
1.0
10.0
100.0
1000.0
Log[cHC84.1 (ng/mL)]
Log[cV3.2 (ng/mL)]
AcꢀC G436 W V A G L F Y Y H K F N448 CꢀNH2
AcꢀC N430 E S L N T G W L A G L F Y Y444 CꢀNH2
AcꢀN430 E S L N T G W L A G L F Y Y444 CꢀNH2
AcꢀG436 W V A G L F Y Y H K F N448 CꢀNH2
15
16
18
17
Figure 4. ELISA evaluating the effect of cyclization by comparing the immobilized cyclic peptides 15 and 17 with the correꢀ
sponding linear epitope mimics 16 and 18 using MAbs HC84.1 and V3.2. (A): MAb HC84.1 (3ꢀfold dilution over by 7 steps [111.1
– 0.2 ng/mL]); (B): MAb V3.2 (3ꢀfold dilution over 7 steps [111.1 – 0.2 ng/mL]). Background signal (no MAb) was subtracted.
The data is represented as a percentage relative to the highest absorbance being set to 100% per individual experiment (A: 15; B:
17). Data was collected from 3 independent experiments, each performed in duplicate. Error bars represent the standard deviation.
Employing epitope mimic coated surfaces for identifi-
cation of key binding residues. Since peptide cyclic peptide
precursor 11 (cp) is elongated at the Nꢀterminus compared to
peptide cyclic peptide precursor 9 (cp), some degree of
crossꢀreactivity of MAbs HC84.1 and V3.2 was expected
towards both cyclic epitope mimics 15 and 17. However, no
such crossꢀreactivity was observed at the antibody concenꢀ
trations used. Interestingly, residues involved in binding
MAb HC84.1 (437W, 441L, 442F, 443Y)9 or MAb V3.2 might
differ. Typically, mutagenesis studies are used to identify
key binding residues within epitopes involved in antibody
recognition. For these, several recombinant mutants of E2
have to be generated that are screened in ELISA to evaluate
the altered binding affinity of the antibodies. The process of
generating a multitude of different recombinant E2 sequencꢀ
es is a rather laborious and timeꢀconsuming process. Instead,
epitope mimics could be used as a more highꢀthroughput
screening method as collections of synthetic peptides can be
easily obtained in a fast and automated fashion. Subsequentꢀ
ly, the different synthetic peptides can be conveniently cyꢀ
clized on linker 7, conjugated on maleimideꢀactivated plates,
and analyzed using ELISA.
In order to evaluate whether cyclic epitope mimics could
be used similar to highꢀthroughput mutagenesis studies for
identification of keyꢀbinding residues, it was decided to try
to identify those involved in binding MAb V3.2. The major
difference in the overlapping peptide sequence between preꢀ
cursor peptides 9 (cp) and 11 (cp) is a ValineꢀtoꢀLeucine
change at position 438. To this end, a mutant precursor pepꢀ
tide 13 (cp) was synthesized that contained a Valineꢀtoꢀ
Leucine change on position 438 (V438L). Cyclic precursor
peptide 13 (cp) was subsequently cyclized to prepare cyclic
epitope mimic 19, which was then conjugated on maleimideꢀ
activated plates, and tested using ELISA. Monoclonal antiꢀ
bodies HC84.1 and V3.2 were tested against plate surfaces
containing immobilized cyclic epitope mimics (15, 17, or
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Bioconjugate Chemistry
cyclic epitope mimic 19 showed a small decrease in binding
19). The cyclic epitope mimic 15 showed good binding of
1
2
3
MAb HC84.1 (Fig. 5A) but no binding of MAb V3.2. In
contrast, cyclic epitope mimic 17 showed good binding of
MAb V3.2 (Fig. 5B). ValineꢀtoꢀLeucine change containing
by MAb HC84.1 (Fig. 5A), but showed improved binding by
MAb V3.2 (Fig. 5B). This result suggested that the leucine
sequence at position 438 is crucial for binding MAb V3.2.
4
5
6
7
8
9
A
B
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
110.0
90.0
70.0
50.0
30.0
10.0
ꢀ10.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.1
1.0
10.0
100.0
0.1
1.0
10.0
100.0
Log[cHC84.1 (ng/mL)]
Log[cV3.2 (ng/mL)]
AcꢀC N430 E S L N T G W L A G L F Y Y444 CꢀNH2
AcꢀC G436 W V A G L F Y Y H K F N448 CꢀNH2
AcꢀC G436 W L A G L F Y Y H K F N448 CꢀNH2
15
19
17
Figure 5. ELISA showing the relative importance of different immobilized cyclic epitope mimics 15, 17, and 19 towards binding
of MAbs HC84.1 and V3.2. (A): MAb HC84.1 (3ꢀfold dilution over 6 steps [37.0 – 0.2 ng/mL]); (B): MAb V3.2 (3ꢀfold dilution
over 5 steps [37.0 – 0.5 ng/mL]). Background signal (no MAb) was subtracted. The data is represented as a percentage relative to
the highest absorbance being set to 100% per individual experiment (A: 15; B: 17). Data was collected from 3 independent experiꢀ
ments, each performed in duplicate. Error bars represent the standard deviation.
CONCLUSION
The novel ELISA platform that has been developed here
provides an easy, straightforward, and reliable strategy for
cyclization of peptides and subsequent conjugation of these
cyclic peptides covalently to an ELISA plate. This opens up
possibilities for studying antibodyꢀepitope interaction in a
highꢀthroughput fashion and might be an attractive tool in
vaccine design.
In conclusion, we have synthesized epitope mimics conꢀ
taining peptides based on Epitope II of the HCV E2 glycoꢀ
protein using benzyl bis bromide derivatives functionalized
with a flexible TEG spacer equipped with a thiolꢀmoiety.
The thiolꢀmoiety enabled chemo selective covalent coating
of epitope mimics to a maleimideꢀactivated surface. As a
result uniform peptide layers were obtained on a solid surꢀ
face of which we hypothesized that the epitope structure is
better preserved than when peptides are nonꢀcovalently abꢀ
sorbed onto the ELISA plates. In addition, covalent adsorpꢀ
tion prevents a gradual leakage of peptides from the surface.
The epitope mimics peptide sequences originated from HCV
E2 and were presented both as cyclic and linear peptides,
allowing investigation of the necessity of conformationally
constraining of peptide sequences for optimal epitope mimꢀ
icry.
Finally, the use of synthetically modified epitope peptides
in which amino acids are replaced instead of using the entire
recombinantly mutated proteins for studying the relative
binding affinity by antibodies employing this approach is
also a particularly promising perspective.
EXPERIMENTAL SECTION
General procedures: All reagents and solvents were used
as received. Fmocꢀamino acids were obtained from Acꢀ
tivotec (Cambridge, United Kingdom) and N,N,N′,N′ꢀ
TetramethylꢀOꢀ(6ꢀchloroꢀ1Hꢀbenzotriazolꢀ1ꢀyl)uranium
hexafluorophosphate (HCTU) was obtained from Matrix
Innovation (Quebec, Canada). Tentagel S RAM resin (partiꢀ
cle size 90ꢁm, capacity 0.25 mmol.gꢀ1) was obtained from
IRIS Biotech (Marktredwitz, Germany). Methyl tert-butyl
ether (MTBE), Hexane (HPLC grade) and TFA were obꢀ
tained from Aldrich (Milwaukee, USA). DMF (Peptide
grade) was obtained from VWR (Lutterworth, United Kingꢀ
dom). Piperidine, DiPEA were obtained from AGTC Biꢀ
oproducts (Hessle, United Kingdom) and 1,2ꢀethanedithiol
(EDT) was obtained from Merck (Darmstadt, Germany).
HPLC grade CH2Cl2 and acetonitrile were obtained from
Fischer Scientific (Loughborough, United Kingdom). Solid
phase peptide synthesis was performed on a PTI TributeꢀUV
peptide synthesizer. Lyophilizations were performed on a
Christ Alpha 2ꢀ4 LDplus apparatus. Reactions were carried
The broadly neutralizing antibody HC84.1 and weakly
neutralizing antibody V3.2 that target epitope II of HCV E2,
successfully recognized the immobilized cyclic epitope mimꢀ
ics. In addition, it has been shown that cyclization was essenꢀ
tial or at least significantly improved recognition of epitope
mimics. Although it is widely assumed that introducing
conformational constraints for example by cyclization in
(synthetic) peptides for effective mimicry of epitopes is cruꢀ
cial or at least beneficial for recognition by antibodies, to our
knowledge, this is the first time that this has been demonꢀ
strated in a convincing molecular experimental approach.
Thus, we do not pretend that our cyclization method will
favor or stabilize a particular secondary structure, but rather
will provide a more stable mimic of the spatial structure of
the peptide segment in the ꢀin this caseꢀ viral envelope proꢀ
tein than is the case when using a linear peptide.
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Page 18 of 23
out at ambient temperature unless stated otherwise. Solvents equiv. for the 0.25 mmol scale) in DMF using the standard
1
2
3
4
5
6
7
8
were evaporated under reduced pressure at 40°C. Reactions
in solution were monitored by TLC analysis and Rfꢀvalues
were determined on Merck preꢀcoated silica gel 60 Fꢀ254
(0.25 mm) plates. Spots were visualized by UVꢀlight and
permanganate stain. Column chromatography was performed
on Siliaflash P60 (40ꢀ63ꢁm) from Silicycle (Canada) or on a
Biotage Isolera One purification system using prepacked
coupling times (described above). In the last step the resin
was washed with DMF (5 x 30 sec), DCM (5 x 30 sec), dried
over a nitrogen flow for 10 min, followed by the cleavage of
the resinꢀbounded peptide. Cleavage and deꢀprotection was
achieved by treatment of the resin with TFA/H2O/TIS/EDT
(15 mL for the 0.25 mmol scale and 5 mL for the 0.1 mmol
scale, 90:5:2.5:2.5, v/v/v/v) for 3 h at rt. The peptide was
precipitated in MTBE/hexane (1:1, 90 mL for the 0.25 mmol
scale and 45 mL for the 0.1 mmol scale), centrifuged, the
supernatant decanted and the pellet washed 3 times with
MTBE/hexane (1:1, 45 mL). The resulting pellet was reꢀ
dissolved in t-BuOH/H2O (1:1, v/v) and lyophilized.
1
silica (KPꢀSIL) Biotage SNAP cartridges. H NMR data was
acquired on a Bruker 400 MHz spectrometer in CDCl3 as
solvent. Chemical shifts (δ) are reported in parts per million
(ppm) relative to trimethylsilane (TMS, 0.00 ppm). Analytiꢀ
cal highꢀpressure liquid chromatography (HPLC) was carried
out on a Shimadzu instrument comprising a communication
module (CBMꢀ20A), autosampler (SILꢀ20HT), pump modꢀ
ules (LCꢀ20AT), UV/Vis detector (SPDꢀ20A) and system
controller (Labsolutions V5.54 SP), with a Phenomenex
Gemini C18 column (110 Å, 5 ꢁm, 250×4.60 mm) or Dr.
Maisch Reprosil Gold 200 C18 (5 ꢁm, 250×4.60 mm). UV
measurements were recorded at 214 and 254 nm, using a
standard protocol: 100% buffer A (acetonitrile/H2O 5:95
with 0.1% TFA) for 2 min followed by a linear gradient of
buffer B (acetonitrile/H2O 95:5 with 0.1% TFA) into buffer
A (0ꢀ100% or 0ꢀ50%) over 30 min at a flow rate of 1.0
mL·minꢀ1. Purification of the peptidic compounds was perꢀ
formed on an Agilent Technologies 1260 infinity preparative
system using both UV and ELSD detectors with a Phenomꢀ
enex Gemini C18 column (110 Å, 10 ꢁm, 250×20 mm) or
Dr. Maisch Reprosil Gold 200 C18 (10 ꢁm, 250×20 mm).
Autoꢀcollection of fractions was used based on the UV
measurements at 214 nm, using a standard protocol: 100%
buffer A for 5 min followed by and linear gradient of buffer
B into buffer A (0ꢀ50% or 20ꢀ60%) over 65 min at a flow
rate of 12.5 mL·minꢀ1 using the same buffers as described for
the analytical HPLC. Liquid chromatography mass specꢀ
trometry (LCMS) was carried out on a Thermo Scientific
LCQ Fleet quadrupole mass spectrometer with a Dionex
Ultimate 3000 LC using a Dr. Maisch Reprosil Gold 120
C18 column (110 Å, 3 ꢁm, 150×4.0 mm) and the same linear
gradients of buffer B into buffer A, flowrate and buffers as
described for the analytical HPLC.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General method for peptide alkylation: Crude precursor
peptide (25 µmol; 1.0 equiv.) was dissolved in DMF (18
mL). Subsequently, a solution of cyclization (6) or linear (7)
linker (37.5 µmol; 1.5 equiv.) in DMF(1 mL) was added.
Subsequently, an aqueous NH4HCO3 buffer solution (pH 7.9,
20 mM; 6 mL) was added dropwise to the reaction mixture,
which gave a general peptide concentration of ≤ 1 mM in
DMF/buffer solution (3:1, v/v). The reaction mixture was
stirred for 2 hours at room temperature, followed by concenꢀ
tration in vacuo. The residue was reꢀdissolved in
TFA/H2O/TIS/EDT (15 mL, 90:5:2.5:2.5, v/v/v/v) and the
resulting reaction mixture was stirred for 3 hours at room
temperature. Then, the crude product was precipitated in 2 x
50 ml MTBE/hexane (1:1, v/v). The crude precipitate was
obtained by centrifugation (4500 rpm, 5 minutes). The colꢀ
lected precipitate was washed an additional four times using
2 x 50 ml MTBE/hexane (1:1, v/v) and centrifugation (4500
rpm, 5 minutes). After this the crude product was dissolved
in tBuOH/H2O (1:1, v/v) and lyophilized.
Monoclonal antibodies: The humanꢀmouse chimeric anꢀ
tibody cHC84.1 was generated by grafting the nucleotide
sequences encoding the variable heavy and variable light
chain of human MAb HC84.1 (10) on the the mouse IgG1
backbone. The resulting chimeric antibody was expressed in
HEKꢀ293T cells and purified using proteinꢀG affinity chroꢀ
matography. The mouse MAb V3.2 was generated by imꢀ
munizing mice with recombinant virusꢀlike particles of
HCV. Like HC84.1, the epitope of V3.2 recognizes amino
acid residues within the epitope II region of HCV E2 glycoꢀ
protein. A detailed characterization of this antibody will be
reported elsewhere.
General method for automated peptide synthesis: The
peptides were synthesized on a PTI TributeꢀUV peptide synꢀ
thesizer. Tentagel S RAM resin (1.0 g, 0.25 mmol, 1.0 equiv
or 400 mg, 0.1 mmol, 1.0 equiv) was allowed to swell in
DMF (3 x 10 min). Deꢀprotection of the Fmoc group was
achieved by treatment of the resin with 20% piperidine in
DMF using the RV_top_UV_Xtend protocol from the Tribꢀ
uteꢀUV peptide synthesizer followed by a DMF washing step
(5 x 30 sec). The Fmocꢀprotected amino acids were coupled
using HCTU (with the 0.1 mmol scale 5 equiv was used and
with the 0.25 mmol scale 4 equiv was used) and DiPEA
(with the 0.1 mmol scale 10 equiv was used and with the
0.25 mmol scale 8 equiv was used) in DMF, as a coupling
system, with 2 min preꢀactivation. The coupling time was 10
min when the peptide was synthesized on a 0.1 mmol scale
and 20 min when the 0.25 mmol scale was conducted. After
every coupling the resin was washed with DMF (6 x 30 sec).
After coupling of the last amino acid, the Fmoc group was
cleaved using the normal deꢀprotection conditions (described
above) and the resulting free Nꢀterminus was acetylated by
treating the resin bounded peptide with acetic anhydride (250
ꢁL) and DiPEA (10 equiv. for the 0.1 mmol scale and 8
General method for ELISA: Pierce® maleimide activatꢀ
ed 96ꢀwell plates were purchased from Thermo Scientific.
The wells were washed three times with 200 ꢁL wash buffer
(0.1 M Na3PO4, 0.15 M NaCl, 0.05% Tween®ꢀ20 detergent;
pH 7.2). Then, the desired epitope mimic (100 – 500 ꢁg) was
suspended in 1 mL binding buffer (0.1 M Na3PO4, 0.15 M
NaCl, 10 mM EDTA; pH 7.2) and further diluted (10 – 50
fold) to a concentration of 10 ꢁg/mL. To each well, 200 ꢁL
of the epitope mimic solution was added and incubated
overnight at 4 °C. After this, the wells were washed three
times with 200 ꢁL wash buffer. For capping unreacted maꢀ
leimide groups, immediately before use, a solution of 10
ꢁg/mL Nꢀacetylated cysteine was prepared and 200 ꢁL was
added to each of the wells, followed by incubation of 1ꢀ2
hours at room temperature. Then, the wells were washed
three times with 200 ꢁL wash buffer. A threeꢀfold dilution
series of primary MAb was prepared and 100 ꢁL was transꢀ
ferred to each well, followed by incubation for 1ꢀ2 hours at
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Bioconjugate Chemistry
Tetraethylene glycol monotrityl thioether (4): All steps
room temperature. After this, the wells were washed three
1
2
3
4
5
6
7
8
times with PBST, before supplying 100 ꢁL 1:2000 secondꢀ
ary HRPꢀconjugated αꢀmouse A4416 (Sigma) antibodies to
each well. Incubation continued for 1ꢀ2 hours at room temꢀ
perature, followed by washing the wells three times with
PBST. The plates were developed using 100 ꢁL 3, 3’, 5, 5’
tetramethylbenzidine (TMB) solution per well, obtained
from Life Technologies, and incubating for 25 minutes at
room temperature, after which, further development was
stopped using 200 ꢁL 0.5 M H2SO4 per well. Absorbance at
450 nm was measured on a Varioskan (Thermoscientific) or
PHERAstar FS (BMG Labtech) instrument.
were performed under N2 atmosphere. Crude triphenylmeꢀ
thylthiol 3 (11.1 g, 40.0 mmol, 2.6 equiv.) was dissolved in
dry THF (100 mL). The resulting solution was cooled to 0
°C using an ice bath, followed by addition of 1.6 g NaH
(60% in mineral oil; 40.0 mmol, 2.6 equiv). The resulting
suspension was stirred for 1 hour at 0 °C. A separate solution
of tetraethylene glycol pꢀtoluenesulfonate 2 (5.4 g, 15.4
mmol, 1.0 equiv.) was prepared in dry THF (10 mL), which
was added to the reaction mixture at 0 °C. After which, the
ice bath was removed and the reaction mixture was stirred
for another 15ꢀ30 minutes at room temperature. Upon comꢀ
pletion of the reaction, as was determined by TLC (EtOAc),
the reaction mixture was quenched by addition of a sat.
NH4Cl (aq) (100 mL) solution while vigorously stirring for
an additional 15 minutes. Then, THF was removed in vacuo
and EtOAc (100 mL) was added. The aqueous and organic
layers were separated. The EtOAc layer was washed with
H2O (2 x 100 mL). The combined aqueous layers were backꢀ
extracted with EtOAc (100 mL). The obtained organic layers
were combined and washed with brine (200 mL), dried over
Na2SO4, and filtered. The filtrate was concentrated in vacuo,
which afforded the crude product as a yellow oil. Purificaꢀ
tion was immediately performed by automated flash column
chromatography (100% EtOAc) afforded tetraethylene glyꢀ
col monotrityl thioether (4) (6.9 g, 15.2 mmol, quant.) as a
yellow oil. Rf = 0.59 (EtOAc); tR = 39.1 min; 1HꢀNMR (400
MHz, CDCl3): δ = 7.42 (m, 6H, trityl oꢀH), 7.28 (m, 6H,
trityl mꢀH), 7.21 (m, 3H, pꢀH), 3.70 (m, 2H, CH2OH), 3.64
(m, 4H, CH2), 3.58 (m, 4H, CH2), 3.45 (m, 2H, CH2), 3.30 (t,
9
10
11
12
13
14
15
16
17
18
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45
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48
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59
60
Tetraethylene glycol ρ-toluenesulfonate (2): All steps
were carried out under N2 atmosphere. Commercially availaꢀ
ble TEG 1 (8.6 ml, 50.0 mmol, 2.0 equiv.) was dissolved in
dry CH2Cl2 (100 mL). The resulting solution was cooled to 0
°C using an ice bath, followed by addition of TEA (7.0 mL,
50.0 mmol, 2.0 equiv.). Then pꢀtoluenesulfonyl chloride (4.8
g, 25 mmol, 1.0 equiv.) was added, the ice bath was removed
and the resulting reaction mixture was stirred for 16 hours at
room temperature. Next, the reaction mixture was washed
with 1 M HCl (aq) (2 x 100 mL) and H2O (100 mL). The
obtained aqueous layers were combined and backꢀextracted
with CH2Cl2 (100 mL). All CH2Cl2 layers were combined
and washed with brine (200 mL) and dried over Na2SO4,
followed by filtration. The filtrate was concentrated in vac-
uo, affording the crude product as a yellowish syrup. Purifiꢀ
cation by automated flash column chromatography (100%
EtOAc) afforded tetraethylene glycol pꢀtoluenesulfonate (2)
(5.4 g, 15.4 mmol, 62%) as a clear yellowish syrup. Rf =
0.45 (EtOAc); tR = 25.0 minutes; 1HꢀNMR (400 MHz,
3
3JHH = 6.9 Hz, 2H, SCH2CH2O), 2.44 (t, JHH = 6.9 Hz, 2H,
SCH2), 2.34 (broad s, 1H, OH) ppm; 13CꢀNMR (101 MHz,
CDCl3): δ = 144.8 (ArꢀC), 129.6 (oꢀArꢀCH), 127.9 (mꢀArꢀ
CH), 126.6 (pꢀArꢀCH), 72.5 (CH2), 70.7 (CH2), 70.5 (CH2),
70.4 (CH2), 70.2 (CH2), 69.7 (SCH2CH2O), 66.6 (SC), 61.8
(CH2), 31.7 (SCH2); HRMS: calculated m/z for
C27H32NaO4S: 475.1914 [M+Na]+1; found 475.1900.
3
CDCl3): δ = 7.79 (d, JHH = 8.2 Hz, 2H, arylꢀH), 7.34 (m,
3
3JHH = 8.2 Hz, 2H, arylꢀH), 4.16 (m, JHH = 4.8 Hz, 2H, Toꢀ
sOCH2), 3.70 (m, 4H, 2 x CH2), 3.64 (m, 2H, 4 x CH2), 3.60
(m, 6H, 3 x CH2), 2.44 (s, 3H, CH3) 2.35 (broad s, 1H, OH)
ppm; 13CꢀNMR (101 MHz, CDCl3): 144.8 (arylꢀC), 133.1
(arylꢀC), 129.8 (mꢀarylꢀCH), 128.0 (oꢀarylꢀCH), 72.5 (CH2),
70.8 (CH2), 70.7 (CH2), 70.5 (CH2), 70.4 (CH2), 69.2 (Toꢀ
sOCH2), 68.7 (CH2), 61.8 (CH2), 21.64 (CH3); HRMS: calꢀ
culated m/z for C15H24NaO7S: 371.1135 [M+Na]+1; found
371.1128.
Benzylic bromide linkers: All steps were carried out unꢀ
der N2 atmosphere. Tetraethylene glycol monotrityl thioether
4 (3.4 g, 7.6 mmol) was dissolved in dry THF (50 mL). The
resulting solution was cooled to 0 °C using an ice bath, folꢀ
lowed by the addition of 0.5 g NaH (60% in mineral oil; 11.4
mmol, 1.5 equiv.). After which, the reaction mixture was
stirred for 1 hour at 0 °C. Then, tetraethylene glycol
monotrityl thioether 4 was added in portions to a solution of
1, 3, 5ꢀtris(bromomethyl)benzene 5 or α, α’ꢀdibromideꢀmꢀ
xylene 6 and stirring continued for 16 hours. Upon compleꢀ
tion of the reaction, as determined by TLC (30% EtOAc in
petroleum ether 40ꢀ60 °C), THF was removed in vacuo.
Then, the residue was dissolved in EtOAc, followed by filꢀ
tration over Celite to remove salts. The filtrate was concenꢀ
trated in vacuo, which afforded the crude product. Purificaꢀ
tion by automated flash column chromatography using a
linear gradient (10ꢀ30% EtOAc in petroleum ether 40ꢀ60 °C
over 20 column volumes) afforded pure product.
Triphenylmethanethiol (3): All steps were carried out
under N2 atmosphere. Commercially available trityl chloride
(13.9 g, 50.0 mmol, 1.0 equiv.) was dissolved in acetone
(200 mL). The resulting solution was added to a vigorously
stirring solution of NaSH.H2O (11.3 g, 201.2 mmol, 4.0
equiv.) in acetone/H2O 1:1, v/v (400 mL). The resulting reꢀ
action mixture was stirred at room temperature for 15
minutes. Then, additional H2O (200 mL) was added to the
reaction mixture and stirring was continued for 1 hour. The
crude product precipitated as a yellow solid, which was filꢀ
tered and was washed thoroughly with H2O. The resulting
yellow solid was dried in vacuo, affording 11.1 g (40.0
mmol, 80%) crude triphenylmethanethiol (3), which was
used without additional purification. Rf
=
0.85
(EtOAc/Petroleum Ether 40ꢀ60 °C 1:9, v/v); tR = 45.6
Cyclization Linker (7): 1, 3, 5ꢀtris(bromomethyl)benzene 5
(1.5 g, 4.2 mmol, 1.1 equiv.) was dissolved in dry THF (75
mL), followed by the portionwise addition (5 x 5 mL) of the
above described reaction mixture containing tetraethylene
glycol monotrityl thioether 4 (1.7 g, 3.8 mmol, 1.0 eq.) with
15 minute intervals. Purification as described above afforded
pure cyclization linker (7) (0.9 g, 1.3 mmol, 33%) as a yelꢀ
low oil. (Rf = 0.31 (petroleum ether 40ꢀ60 °C/EtOAc 7:3,
1
minutes; H NMR (400 MHz, CDCl3): δ = 7.26 (m, 15H,
arylꢀH), 3.08 (s, 1H, SH) ppm; 13C NMR (101 MHz, CDCl3):
δ = 147.2 (arylꢀC), 129.3 (arylꢀCH), 127.8 (arylꢀCH), 126.9
(arylꢀCH), 62.9 (CS) ppm. HRMS: calculated m/z for
C19H15S: 275.0900 [MꢀH]ꢀ; found 275.0892.
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Bioconjugate Chemistry
Page 20 of 23
1
v/v); tR = 49.1 minutes; H NMR (400 MHz, CDCl3): δ =
7.41 (m, 6H, oꢀtritylꢀCH), 7.32 (m, 1H, pꢀarylꢀCH), 7.29 (m,
2H, oꢀarylꢀCH), 7.27 (m, 6H, mꢀtritylꢀCH), 7.21 (m, 3H, pꢀ
tritylꢀCH), 4.53 (s, 2H, OCH2ꢀaryl), 4.45 (s, 4H, 2 x CH2Br),
3.66 (m, 8H, 4 x CH2), 3.58 (m, 2H, CH2), 3.45 (m, 2H,
Cyclic precursor peptide 13 (cp): The peptide was synꢀ
1
2
3
4
thesized as described above by solid phase synthesis on a
0.25 mmol scale, which afforded crude peptide (382.7 mg).
This crude peptide was used in the cyclization by alkylation
step. tR = 19.7 minutes; m/z calculated for C90H119N21O19S2:
931.93 ½[M+2H]2+; found: 932.33.
3
3
CH2), 3.30 (t, JHH = 6.9 Hz, 2H, SCH2CH2), 2.43(t, JHH
=
5
6
7
8
6.9 Hz, 2H, SCH2) ppm; 13C NMR (101 MHz, CDCl3): δ =
144.8 (tritylꢀC), 139.9 (arylꢀC(CH)2), 138.5 (arylꢀ
C(CH2Br)2), 129.6 (oꢀtritylꢀCH), 128.8 (arylꢀCH(CCH2Br)2),
128.1 (oꢀarylꢀCH), 127.9 (mꢀtritylꢀCH), 126.6 (pꢀtritylꢀCH),
72.5 (OCH2ꢀaryl), 70.7 (2 x CH2), 70.6 (CH2), 70.5 (CH2),
70.2 (CH2), 69.9 (CH2), 69.6 (SCH2CH2), 66.6 (CS), 32.8
(CH2Br), 31.7 (SCH2) ppm; HRMS: calculated m/z for
C36H40NaO4S79Br2: 749.0906 [M+Na]+1; found 749.0886.
Cyclic precursor peptide 14 (cp): The peptide was synꢀ
thesized as described above by solid phase synthesis on a
0.25 mmol scale, which afforded crude peptide (332.3 mg).
This crude peptide was used in the cyclization by alkylation
step. tR = 19.1 minutes; m/z calculated for C90H119N21O19S2:
931.93 ½[M+2H]2+; found: 932.50 (13C [+1]).
9
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Cyclic epitope mimic 15: crude cyclic precursor 9 (cp)
(46.2 mg, 25 ꢁmol, 1.0 equiv.) was treated with cyclization
linker 7 (27.4 mg, 38 ꢁmol, 1.5 equiv.) according to the genꢀ
eral method for peptide alkylation as described above. The
obtained crude product (53.9 mg) was dissolved in HPLC
buffers A and B (1:1, v/v) as three batches of 10ꢀ20 mg in 3
ml. Each batch was purified by preparative HPLC using a
linear gradient of HPLC buffer B in HPLC buffer A (0ꢀ50%;
60 minutes). Fractions containing pure product were comꢀ
bined and lyophilized to yield pure cyclic epitope mimic 15
(9.7 mg, 15.4% overall yield; 95% average yield per step) as
a fluffy white powder. tR = 21.6 minutes; LRMS: calculated
m/z for C106H141N21O23S3: 1087.00 ½[M+2H]+2; found
1087.50 (13C [+1]); HRMS: calculated m/z for
C106H141N21O23S3: 1086.9914 ½[M+2H]+2; found 1086.9400.
Linear Linker (8): α, α’ꢀdibromideꢀmꢀxylene 6 (1.1 g, 4.2
mmol, 1.1 equiv.) was dissolved in dry THF (75 mL), folꢀ
lowed by the portionwise addition (5 x 5 mL) of the above
described reaction mixture containing tetraethylene glycol
monotrityl thioether 4 (1.7 g, 3.8 mmol, 1.0 eq.) with 15
minute intervals. Purification as described above afforded
pure cyclization linker (8) (1.0 g, 1.6 mmol, 43%) as a yelꢀ
low oil Rf = 0.36 (petroleum ether 40ꢀ60 °C/EtOAc 7:3, v/v);
tR = 48.3 minutes; 1H NMR (400 MHz, CDCl3): δ = 7.41 (m,
6H, oꢀtritylꢀCH), 7.37 (m, 1H, pꢀarylꢀCH), 7.30 (m, 3H, oꢀ
arylꢀCH), 7.27 (m, 6H, mꢀtritylꢀCH), 7.21 (m, 3H, pꢀtritylꢀ
CH), 4.54 (s, 2H, OCH2ꢀaryl), 4.48 (s, 4H, 2 x CH2Br), 3.64
(m, 8H, 4 x CH2), 3.57 (m, 2H, CH2), 3.45 (m, 2H, CH2),
3
3
3.30 (t, JHH = 6.9 Hz, 2H, SCH2CH2), 2.42 =(t, JHH = 6.9
Hz, 2H, SCH2) ppm; 13C NMR (101 MHz, CDCl3): δ =
144.8 (tritylꢀC), 139.1 (arylꢀC(CH)2), 137.9 (arylꢀCCH2Br),
129.6 (oꢀtritylꢀCH), 128.8 (arylꢀCCH(CCH2Br)), 128.2 (arylꢀ
CH), 127.9 (mꢀtritylꢀCH), 127.7 (arylꢀCH), 126.6 (pꢀtritylꢀ
CH), 72.8 (OCH2ꢀaryl), 70.7 (3 x CH2), 70.5 (CH2), 70.2
(CH2), 69.7 (CH2), 69.6 (SCH2CH2), 66.6 (CS), 33.4
(CH2Br), 31.7 (SCH2) ppm; HRMS: calculated m/z for
C35H39NaO4S79Br: 657.1645 [M+Na]+1; found 657.1629.
Linear epitope mimic 16: crude linear precursor 10 (lp)
(66.4 mg, 38 ꢁmol, 1.0 equiv.) was treated with linear linker
8 (35.9 mg, 57 ꢁmol, 1.5 equiv.) according to the general
method for peptide alkylation as described above. The obꢀ
tained crude product (67.8 mg) was dissolved in HPLC buffꢀ
ers A and B (1:1, v/v) as two batches of 20ꢀ40 mg in 2ꢀ5 ml.
Each batch was purified by preparative HPLC using a linear
gradient of HPLC buffer B in HPLC buffer A (0ꢀ50%; 60
minutes). Fractions containing pure product were combined
and lyophilized to yield pure linear epitope mimic 16 (23.1
mg, 29.3% overall yield; 96% average yield per step) as a
fluffy white powder. tR = 22.1 min; LRMS: calculated m/z
for C102H136N20O22S2: 1029.49 ½[M+2H]+2; found 1030.00
(13C [+1]); HRMS: calculated m/z for C102H136N20O22S2:
1029.4868 ½[M+2H]+2; found 1029.4826.
Cyclic precursor peptide 9 (cp) The peptide was syntheꢀ
sized as described above by solid phase synthesis on a 0.25
mmol scale, which afforded crude peptide (397.9 mg). This
crude peptide was used in the cyclization by alkylation step.
tR
=
19.2 minutes; LRMS: m/z calculated for
C89H117N21O19S2: 924.92 ½[M+2H]2+; found: 925.33 (13C
[+1]).
Cyclic epitope mimic 17: crude cyclic precursor 11 (cp)
(50.5 mg, 25 ꢁmol, 1.0 equiv.) was treated with cyclization
linker 7 (27.7 mg, 38 ꢁmol, 1.5 equiv.) according to the genꢀ
eral method for peptide alkylation as described above. The
obtained crude product (60.7 mg) was dissolved in HPLC
buffers A and B (1:1, v/v) as three batches of respectively
15ꢀ25 mg in 3 ml. Each batch was purified by preparative
HPLC using a linear gradient of HPLC buffer B in HPLC
buffer A (20ꢀ60%; 60 minutes). Fractions containing pure
product were combined and lyophilized to yield pure cyclic
epitope mimic 17 (4.3 mg, 4.7% overall yield; 93% average
yield per step) as a fluffy white powder. tR = 22.6 min;
LRMS: calculated m/z for C108H151N21O30S3: 1160.01
½[M+2H]+2; found 1160.42 (13C [+1]); HRMS: calculated
m/z for C108H151N21O30S3: 1157.9971 ½[Mꢀ2H]+2; found
1157.9971.
Linear precursor peptide 10 (lp): The peptide was synꢀ
thesized as described above by solid phase synthesis on a
0.25 mmol scale, which afforded crude peptide (433.5 mg).
This crude peptide was used in the alkylation step. tR = 18.9
minutes; LRMS: m/z calculated for C86H112N20O18S: 873.42
½[M+2H]2+; found: 873.92 (13C [+1]).
Cyclic precursor peptide 11 (cp): The peptide was synꢀ
thesized as described above by solid phase synthesis on a
0.25 mmol scale, which afforded crude peptide (322.3 mg).
This crude peptide was used in the cyclization by alkylation
step. tR = 21.7 minutes; m/z calculated for C91H127N21O26S2:
997.94 ½[M+2H]2+; found: 998.08 (13C [+1]).
Linear precursor peptide 12 (lp): The peptide was synꢀ
thesized as described above by solid phase synthesis on a
0.25 mmol scale, which afforded crude peptide (390.5 mg).
This crude peptide was used in the alkylation step. tR = 21.5
minutes; m/z calculated for C88H122N20O25S: 946.44
½[M+2H]2+; found: 946.75 (13C [+1]).
Linear epitope mimic 18: crude linear precursor 12 (lp)
(72.6 mg, 38 ꢁmol, 1.0 equiv.) was treated with linear linker
8 (35.9 mg, 57 ꢁmol, 1.5 equiv.) according to the general
method for peptide alkylation as described above. The obꢀ
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Bioconjugate Chemistry
Author Contributions
tained crude product (77.6 mg) was dissolved in HPLC buffꢀ
1
2
3
4
5
6
7
8
ers A and B (1:1, v/v) as two batches of 20ꢀ30 mg in 2ꢀ3 ml.
Each batch was purified by preparative HPLC using a linear
gradient of HPLC buffer B in HPLC buffer A (20ꢀ60%; 60
minutes). Fractions containing pure product were combined
and lyophilized to yield pure linear epitope mimic 18 (3.5
mg, 3.4% overall yield; 91% average yield per step) as a
fluffy white powder. tR = 23.6 min; LRMS: calculated m/z
for C104H146N20O29S2: 1102.51 ½[M+2H]+2; found 1102.75
(13C [+1]). HRMS: calculated m/z for C104H146N20O29S2:
1102.5082 ½[M+2H]+2; not found (no ionization).
The manuscript was written through contributions of all authors.
Acknowledgements
We are grateful to Dr. Mohammed Hezwani, Dr. Susan Ganꢀ
non, and Dr. Alex Hoose for their invaluable support and
helpful discussions during this study.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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52
53
54
55
56
57
58
59
60
Notes
Cyclic epitope mimic 19: crude cyclic precursor 13 (cp)
(46.4 mg, 25 ꢁmol, 1.0 equiv.) was treated with cyclization
linker 7 (27.3 mg, 38 ꢁmol, 1.5 equiv.) according to the genꢀ
eral method for peptide alkylation as described above. The
obtained crude product (51.2 mg) was dissolved in HPLC
buffers A and B (1:1, v/v) as three batches of 10ꢀ20 mg in 2ꢀ
3 ml. Each batch was purified by preparative HPLC using a
linear gradient of HPLC buffer B in HPLC buffer A (0ꢀ50%;
60 minutes). Fractions containing pure product were comꢀ
bined and lyophilized to yield pure cyclic epitope mimic 19
(4.9 mg, 7.4% overall yield; 93% average yield per step) as a
fluffy white powder. tR = 22.3 min; LRMS: calculated m/z
for C107H143N21O23S3: 1094.00 ½[M+2H]+2; found 1094.50
(13C [+1]); HRMS: calculated m/z for C107H143N21O23S3:
1093.9993 ½[M+2H]+2; found 1093.9941.
The authors declare no competing financial interest
Funding Sources
Work in AHP’s laboratory is supported by the MRC Core fundꢀ
ing (MC_UU 12014/2).
ABBREVIATIONS
cp, cyclic precursor; ELISA, enzymeꢀlinked immunosorbent
assay; HCV, Hepatitis C Virus; HPLC, high performance liquid
chromatography; HRMS, high resolution mass spectrometry; lp,
linear precursor; LRMS, low resolution mass spectrometry;
Mab, monoclonal antibody; NAb, neutralizing antibody; PBST,
phosphateꢀbuffered saline/tween; TEG, tetraethylene glycol;
TLC, thin layer chromatography.
Cyclic epitope mimic 20: crude cyclic precursor 14 (cp)
(46.4 mg, 25 ꢁmol, 1.0 equiv.) was treated with cyclization
linker 7 (27.3 mg, 38 ꢁmol, 1.5 equiv.) according to the genꢀ
eral method for peptide alkylation as described above. The
obtained crude product (50.0 mg) was dissolved in HPLC
buffers A and B (1:1, v/v) as three batches of 10ꢀ20 mg in 2ꢀ
3 ml. Each batch was purified by preparative HPLC using a
linear gradient of HPLC buffer B in HPLC buffer A (0ꢀ50%;
60 minutes). Fractions containing pure product were comꢀ
bined and lyophilized to yield pure cyclic epitope mimic 20
(6.7 mg, 8.8% overall yield; 93% average yield per step) as a
fluffy white powder. tR = 21.8 min; LRMS: calculated m/z
for C107H143N21O23S3: 1094.00 ½[M+2H]+2; found 1094.50
(13C [+1]); HRMS: calculated m/z for C107H143N21O23S3:
1093.9992 ½[M+2H]+2; found 1093.9936.
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6. Tarr, A. W., Khera, T., Hueging, K., Sheldon, J.,
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ASSOCIATED CONTENT
Supporting Information
•
1HꢀNMR and 13CꢀNMR, including COSY, 13CꢀDEPT,
and HSQC spectra for compounds 2, 3, 4, 7, and 8;
Analytical HPLC chromatograms of compounds 2, 3,
4, 7, 8 and 15-20; Including a chromatogram overlay
of fractions taken after purification by preparative
HPLC that afforded purified compounds 15ꢀ20;
LRMS and/or HRMS analysis for compounds 2, 3, 4,
7, 8 and 15ꢀ20;
•
•
The Supporting Information is available free of charge on the
ACS Publications website.
8. Sautto, G., Tarr, A. W., Mancini, N., Clementi, M.
(2013) Structural and antigenic definition of hepatitis
C virus E2 glycoprotein epitopes targeted by monoꢀ
clonal antibodies. Clin. Dev. Immunol. 2013. 450963.
9. Keck, Z. Y., Xia, J., Wang, Y., Wang, W., Krey, T.,
Prentoe, J., Carlsen, T., Li, A. Y., Patel, A. H., Lemon,
S. M., et al. (2012) Human monoclonal antibodies to a
The file will be available as PDF.
AUTHOR INFORMATION
Corresponding Author
* RMJL (robert.liskamp@glasgow.ac.uk) or AHP
(arvind.patel@glasgow.ac.uk)
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Page 22 of 23
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