Double conjugation strategy to incorporate lipid adjuvants into multiantigenic vaccines

Article abstract of DOI:10.1039/c5sc03859f

Conjugation of multiple peptides by their N-termini is a promising technique to produce branched multiantigenic vaccines. We established a double conjugation strategy that combines a mercapto-acryloyl Michael addition and a copper-catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction to synthesise self-adjuvanting branched multiantigenic vaccine candidates. These vaccine candidates aim to treat cervical cancer and include two HPV-16 derived epitopes and a novel self-adjuvanting moiety. This is the first report of mercapto-acryloyl conjugation applied to the hetero conjugation of two unprotected peptides by their N-termini followed by a CuAAC reaction to conjugate a novel synthetic lipoalkyne self-adjuvanting moiety. In vivo experiments showed that the most promising vaccine candidate completely eradicated tumours in 46% of the mice (6 out of 13 mice).

Full text of DOI:10.1039/c5sc03859f

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DOI: 10.1039/C5SC03859F.
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Double Conjugation Strategy to Incorporate Lipid Adjuvants into
Mulꢀanꢀgenic Vaccines†
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
a
c
Waleed M. Hussein, Tzu-Yu Liu, Pirashanthini Maruthayanar, Saori Mukaida, Peter M. Moyle,
b
a,c,d
a
James W Wells, Istvan Toth,*
and Mariusz Skwarczynski*
DOI: 10.1039/x0xx00000x
Conjugation of multiple peptides by their N-termini is a promising technique to produce branched multiantigenic vaccines.
We established a double conjugation strategy that combines a mercapto-acryloyl Michael addition and a copper-catalysed
alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction to synthesise self-adjuvanting branched multiantigenic vaccine
candidates. These vaccine candidates aim to treat cervical cancer and include two HPV-16 derived epitopes and a novel
self-adjuvanting moiety. This is the first report of mercapto-acryloyl conjugation applied to the hetero conjugation of two
unprotected peptides by their N-termini followed by a CuAAC reaction to conjugate a novel synthetic lipoalkyne self-
adjuvanting moiety. In vivo experiments showed that the most promising vaccine candidate completely eradicated
tumours in 46% of the mice (6 out of 13 mice).
www.rsc.org/
common cancer among women. According to experimental and
epidemiological studies, human papilloma virus (HPV) is the main
cause of cervical cancer. Two high-risk genotypes, HPV types 16
Introduction
7
The ability to develop safe vaccines using minimal microbial
components has triggered rapid growth in research into peptide-
(
HPV-16) and 18 (HPV-18) are responsible for 70% of all cervical
8
cancers.
Prophylactic vaccines against HPV infection help to reduce the
1
based vaccines. However, the inability of peptides in isolation to
stimulate the immune system is one of the key challenges in the
development of peptide-based vaccines. Therefore, an adjuvant
incidence of cervical cancers through the generation of neutralizing
antibodies and are only effective if administered before infection
(
immunostimulant) is necessary to stimulate a potent immune
9
with HPV. Hence, there is a strong demand for the development of
2
response against peptide epitopes. However, the use of adjuvants
is usually associated with side effects and substantial toxicity that
has limited the number of adjuvants approved for human use. Only
alum has been approved as a general human adjuvant, while just a
few others were approved for particular vaccine formulation e.g.
MF59, ASO3 and ASO4. Unfortunately for anticancer vaccines,
effective therapeutic vaccines that are able to treat HPV-related
10
cancers. The HPV genome encodes two types of proteins: early
3
proteins (E1, E2, E4, E5, E6 and E7) and late proteins (L1 and L2).
Expression of the E6 and E7 oncoproteins results in deregulation of
the cell cycle, inactivating tumour suppressor gene products p53
4
10
and retinoblastoma protein (pRb) and leading to cancer.
adjuvants that stimulate safe and effective cytotoxic T lymphocyte
Peptide-based strategies to develop therapeutic vaccines against
HPV-associated cancers have shown promising outcomes in several
5
(
CTL) responses are scarce. To overcome this problem, peptide
vaccine research has turned its focus to the development of self-
adjuvanting delivery systems. These vaccines combine peptide
epitopes and immunostimulatory moieties (for example lipidic or
polymeric entities) in a single covalently-linked conjugate, thereby
ensuring co-delivery of the antigen to antigen presenting cells
10
early stage clinical trials. The choice of an appropriate peptide
antigen is a crucial issue in the design of synthetic peptide vaccines.
Therapeutic vaccines to treat cancer must elicit cellular immunity,
+
1a
thus must include CTL (CD8 ) epitopes. A CTL epitope was
identified in the HPV-16 E6 protein sequence (QLLRREVYDFAFRDL;
(
APCs) activated by immunostimulatory moieties. This combined
10-11
)
11-12
E643-57
and was previously shown to induce CTLs in vivo.
presentation helps to enhance vaccine potency and to avoid
undesirable side effects that result from using classical adjuvants.
Every year, approximately 500,000 women are newly diagnosed
with cervical cancer around the world, making it the second most
Recently, our group showed that the 8Qmin peptide, a small
6
9
fragment of HPV-16 E7 protein (QAEPDRAHYNIVTF; E744-57), that
encodes CTL and T-helper cell epitopes, could reduce tumour
growth and eradicate E7-expressing TC-1 tumour cells in mice
1
3
through activation of CTLs when administered with a self-
adjuvanting delivery system. Therefore, these two CTL epitopes,
E643-57 and 8Qmin, were chosen as promising antigens for peptide
vaccine development. We also recently demonstrated that anti-
8
Q
min antibodies were not produced by mice vaccinated with the
8
Qmin epitope conjugated to the poly tert-butyl acrylate delivery
1
4
system.
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Scheme 1 (a) Comparison between the structure of Pam2Cys and
the lipoalkynes 1-3: (i) hydrolyzable ester linkages in Pam2Cys
versus stable ether bonds in lipoalkynes 1-3, (ii) two chiral centers
in Pam2Cys versus achiral molecule in lipoalkynes 1-3, and (iii)
presence of alkyne moiety in lipoalkynes 1-3. (b) Synthesis of the
a
lipoalkyne vaccine adjuvants 1-3. Compound 8 is a commercially
available compound.
Fig.
1
Optimizing the conditions for mercapto-acryloyl
conjugation between model mercapto-azide (12) that used in
excess and acryloyl derivative (13) peptides (a) at 0 time; (b) at
3 h, using DMF as a solvent and two drops of DIPEA. New
products started to form including the mercapto-acryloyl
conjugation product (14) and the dimer of 12; (c) at 7 h in
DMF/DIPEA the reaction was completed with the formation of
peptide 14 and the dimer of 12 as major products with the
remaining of the majority of the acryloyl peptide 13; (d) at 3 h,
using guanidine buffer as a solvent (~pH 7.3) at 37 °C, new
products formed, the mercapto-acryloyl conjugation product
It was reported that the orientation of antigens in a vaccine
conjugate was very important for stimulating an immune
1
5
response. Conjugation of different peptides via the C-terminus is
valuable for the development of multiantigenic branched vaccines.
1
6
Branched antigens tend to have increased stability to proteolysis,
and therefore a longer circulation time in the host, providing more
opportunities to be taken up by APCs. As a result, these peptides
1
7
can elicit stronger in vivo immune responses than linear peptides.
We recently reported that modification of the 8Qmin epitope from
the E7 protein by replacing the C-terminal CCKCD sequence with
SSKSD or SKKKK substantially diminished its immunogenicity. In
contrast, deletion of the CCKCD sequence did not have any negative
(
14) together with the dimer of 12; (e) at 14 h in a guanidine
buffer, the reaction was completed by formation of peptide 14
as a major product together with the dimer of 12 and
complete consumption of the acryloyl peptide 13. The reaction
progress was monitored by HPLC and the products were
detected by mass spectrometry.
9
influence on the epitope potency. These results suggest that the
CTL epitope is only effective if conjugated to the vaccine delivery
system via its N-terminus.
Fig.
alkyne−azide
2
A
copper-catalysed
1,3-dipolar
cycloaddition (CuAAC) model
reaction between model
conjugation product (14) and
model alkyne (15) in DMF in
presence of Cu wire at 50 °C
under a nitrogen atmosphere
(
a) at 0 time; (b) at 1 h the
reaction was completed by the
complete consumption of 14
and formation of the model
CuAAC product 16.
Scheme 2 Model double conjugation through a Michael addition,
between 12 and 13, followed by a CuAAC reaction, between 14 and
1
5.
2
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Scheme 3 One pot reaction of model double conjugation through
mercapto-acryloyl reaction, between 17 and 18 model peptides,
followed by azide-alkyne reaction, between compounds 19 and 15.
Scheme 4 Synthesis of lipopeptides 24-26 using the double
conjugation strategy.
The attachment of a lipidic moiety to the N-terminus of an antigenic
peptide to obtain amphiphilic vaccine molecules was previously
Results and Discussion
6
c, 6d, 18
reported.
However, the N-terminal conjugation of two or We designed and synthesised immunostimulatory lipoalkynes 1-3
more different unprotected epitopes to a vaccine delivery system (Scheme 1a). These lipoalkynes were based on the structure of
have not yet been described (to the best of our knowledge). Thus Pam2Cys (di-palmitoyl-S-glycerol cysteine), well-characterised
we established a double conjugation synthetic technique to allow self-adjuvanting moiety that is widely used in experimental vaccine
a
1
9
the conjugation of different unprotected peptides, E643-57 and design. As Pam2Cys is a thio-1,2-diglyceride ester of palmitic acid,
min, via their N-termini in order to produce novel branched the new 1,3-diglyceride lipoalkynes 1-3 were designed by replacing
8
Q
multiantigenic immunotherapeutics.
the two ester linkages in Pam2Cys with two ether bonds to increase
the stability of the compounds against esterases. The two long
hydrocarbon chains in Pam2Cys were modified by substituting two
methylene groups with oxygen atoms in two different positions as
in lipid 1 and 2, to investigate the effect of increasing the polarity
(
and subsequently the aqueous solubility) on the adjuvanting effect
2
0
of the resulting molecules. For control purposes, lipid 3 contained
the same hydrocarbon chain as Pam2Cys was synthesised. In
contrast to Pam2Cys, lipids 1-3 have no chiral center and therefore
exist as single isomers. They carry an alkyne moiety, thereby
allowing easy conjugation of an antigen through a copper-catalysed
alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction.
Lipoalkynes 1-2 were synthesised using three straightforward
steps (Scheme 1b), while lipoalkyne 3 required only two steps to be
produced. Alcohols 6-7 were prepared from diols 4-5 in 42 and 44%
yields, respectively, using alkyl bromide and a phase-transfer
Fig.
conjugation between mercapto-
Fig. 3 One pot double conjugation model reaction. Mercapto- azide (21) and acryloyl
4
Mercapto-acryloyl
acryloyl conjugation between mercapto-azide (17) and acryloyl derivative (22) at pH 7.5, 37 °C
derivative (18) at ~pH 7.3 (guanidine buffer), 37 °C (a) at 0 time; (b) (a) at 0 time; (b) at 48 h the
at 72 h the reaction was completed by the complete consumption reaction was completed by the
of 17 and formation of the conjugation product (19) together with complete consumption of 22
the disulfide dimer of 17. In a one pot reaction, a CuAAC model and
formation
of
the
reaction between model conjugation product (19) and model multiantigenic
conjugation
alkyne (15) in guanidine buffer in presence of Cu wire at 50 °C product (23) together with the
under a nitrogen atmosphere (c) at 0 time; (d) at 1 h the reaction dimer of 21. The reaction
was completed by the complete consumption of 19 and formation progress was monitored by
of the model CuAAC product 20. The reaction progress was HPLC and the products were
monitored by HPLC and the products were detected by Mass spec.
detected by MS.
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Scheme 5 Synthesis of 27 (lipid 1 conjugated with 8Qmin) and 28
lipid 1 conjugated with E643-57).
(
catalyst tetrabutylammonium iodide (TBAI) in presence of DMF as a
solvent under sonication conditions. The sonication of a mixture of
alcohols 6-8, powdered sodium hydroxide, epichlorohydrin, and
tetrabutylammonium bromide (TBABr) provided the branched
alcohols 9-11. Alcohols 9-11 were treated with propargyl bromide
and sodium hydride to afford the lipoalkynes 1-3 in good yields.
A
double conjugation strategy was developed to produce
anticancer vaccine candidates. Two peptide epitopes were
combined into a multiantigenic construct via thioether conjugation
using an acryloyl peptide. The reaction was examined on two model
short peptides (12 and 13), where one peptide carried both
mercapto and azide groups at its N-terminus (12) and the other
peptide had an acryloyl moiety attached to its N-terminus (13)
(
Scheme 2).
Fig. 5 Tumor challenge experiments. C57BL/6 (8/group) were
5
inoculated subcutaneously in the right flank with 1 × 10 TC-1 cells
/mouse (day 0) and immunised with: (a) lipopeptides 24-26; a
physical mixture of lipid 1 conjugated with 8Qmin (27) and lipid 1
conjugated with E643-57 (28); 8Qmin/E643-57 epitopes conjugated with
Pam2Cys (29) as a positive control; or PBS as a negative control on
day 3 (8/group) or (b) lipopeptide 24; a mixture of 8Qmin-E643-57 (1:1)
+
ISA51 as a positive control; or PBS as a negative control on day 3
(
13/group). Survival rate was monitored over 60 days post
implantation and plotted as a Kaplan−Meier survival curve. Mice
3
were euthanised when tumor volume reached 1 cm or started
bleeding. The survival rate of each group was compared to the
negative control (PBS) and was analysed using the log-rank (Mantel-
Cox) test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
The mercapto-acryloyl conjugation conditions were optimised;
~pH 7.3, 37 °C, 14 h in the presence of denaturants (6 M guanidine)
was found to be optimal. The product 14 of this conjugation was
reacted with a model alkyne 15 producing the desired conjugate 16
(
Scheme 2, Fig. 1 and 2). The ability of double conjugation strategy
to be performed in a one pot reaction was also demonstrated.
(
Scheme 3, Fig. 3).
The new vaccine candidates, lipopeptides 24-26, were
synthesised using the developed conjugation method. First, the N-
terminal amine moieties of 8Qmin and E643-57 were modified using
stepwise SPPS. Fmoc-cysteine and azidoacetic acid were coupled to
8
(
2
Q
min to produce mercapto-azide derivative 21. The second peptide
E643-57) was modified with acrylic acid to afford acrylate derivative
2. The two modified unprotected peptides (21 and 22) were then
Scheme 6 Synthesis of 8Qmin/E643-57 -Pam2Cys (29).
conjugated via a Michael addition mercapto-acrylate reaction to
produce azide derivative 23.
4
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5
Figure 6. Tumor challenge experiments. (a) C57BL/6 (8 per group) were inoculated subcutaneously in the right flank with 1 × 10 TC-1
tumour cells /mouse (day 0) and immunised with lipopeptide 24, a mixture of lipid 1 conjugated with 8Qmin (27) and lipid 1 conjugated
with E643-57 (28) (1:1), 8Qmin/E643-57-Pam2Cys (29) as a positive control, or PBS as a negative control on day 3. (b) C57BL/6 mice (13 per
5
group) were inoculated subcutaneously in the right flank with 1 × 10 TC-1 tumour cells /mouse (day 0) and immunised with lipopeptide
2
different groups of mice shown up to day 24 after tumour implantation (when the first mouse was euthanised). (c) Tumour volume (cm )
3
4, a mixture of 8Qmin + E643-57 (1:1) + ISA51 as a positive control, or PBS as a negative control on day 3. Mean tumour volume (cm ) in
3
in individual TC-1 tumour bearing mice (C57BL/6 mice, 13 per group) treated with lipopeptide 24 or (d) 8Qmin + E643-57 + ISA51 shown
over 60 days post implantation. Mean tumour volume (cm ) in different groups of mice (C57BL/6 mice, 13 per group) shown up to day 24
3
+
Fig. 7 Assessment of CD8 T-cell response to vaccination. Mice were immunised with lipopeptide 24, a mixture of E643-57 and 8Qmin
(
E6/E7), an “irrelevant” lipopeptide (KQAEDKVKASREAKKQVEKALEQLEDKVK - conjugated with lipid 1), or PBS subcutaneously in both
flanks. Ten days later, spleens were harvested and IFN-γ production in response to (a) short E6 (YDFAFRDL) or (b) short E7 (RAHYNIVTF)
peptides was determined by ELISPOT (n=9 mice/group). The data were pooled from two independent experiments and analysed using
the unpaired t test (*p < 0.05; ***p < 0.001).
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A solution of mercapto-azide 21 (2 equiv.) and the acryloyl compounds 24 and 25 formed particles of submicron size (0.3-
derivative 22 (1 equiv.) were gently shaken in denaturing buffer 0.8 µm as measured by dynamic light scattering) under aqueous
comprised of 6 M guanidine, 50 mM sodium phosphate, 20% conditions. This observation is in the agreement with well-known
acetonitrile, 5 mM EDTA, at ~pH 7.3 to afford the azide derivative phenomena that the immune responses are highly dependent on
2
2
3 in an excellent isolated yield of 90% (Scheme 4). The reaction the vaccine particle size. This size difference may have arisen
was monitored by analytical HPLC and mass spectroscopy (Fig. 4). because the presence of oxygen atoms in the hydrocarbon chain in
The second conjugation between the azide derivative 23 (1 equiv.) both lipids 1 and 2 increased the solubility of the latter compounds
and the lipoalkynes 1-3 (1.5 equiv.) was achieved in degassed DMF (24 and 25).
under a nitrogen atmosphere using the CuAAC reaction in the
Compound 29 (which bore a Pam2Cys moiety) induced
6b
presence of copper wire for 4 hours at 50 °C to produce the final unexpectedly weak antitumour responses, therefore two additional
lipopeptides 24-26 in 49-87% isolated yields (Scheme 4). The final independent experiments (with 5 + 8 mice per group) were
products 24-26 were structurally well-defined, with only one performed to further investigate the antitumour potency of the
stereoisomer present, and the synthesis was simple and lead vaccine candidate. Incomplete Freund's adjuvant (Montanide
reproducible.
The therapeutic effect of the multiantigenic conjugates on E643-57 epitopes for formulation of the positive control.
established HPV tumour was evaluated in a mouse model, 6-8 week
Female C57BL/6 (6−8 weeks old) mice (5 + 8 /group) were
ISA51) was chosen as an adjuvant in an emulsion with 8Qmin and
old, female C57BL/6 mice. The design of compounds 1-3 was based immunized with either lipopeptide 24 (100 μg/100 μL sterile PBS),
on the structure of Pam2Cys, hence Pam2Cys conjugated to the 30 μg of a mixture of 8Qmin and E643-57 emulsified in a total volume
8Qmin/E643-57 epitopes was used as a control (29) (Scheme 6). The of 100 μL of incomplete Freund's adjuvant (Montanide ISA51)/PBS
therapeutic importance of incorporating two epitopes in one (1:1, v/v) as a positive control. The Kaplan−Meier survival curve
molecular entity (24-26) was explored by synthesising compounds (Figure 5b) showed that 46% (6 out of 13 mice) of mice treated with
2
7 and 28, which were comprised of lipid 1 conjugated to 8Qmin or lipopeptide 24 survived over 60 days. Vaccination with lipopeptide
E643-57, respectively. At day zero, mice (8/group) were implanted in 24 induced significantly better survival in tumour-bearing mice than
the side flank with TC-1 tumour cells expressing the E6/E7 treatment with the PBS as a negative control (p = 0.0002) (Figure
2
1
oncoproteins. On day
3 mice were immunised with either 5b). As shown in Figure 6b, tumour-bearing mice treated with
lipopeptides 24-26 (100 μg/100 μL sterile PBS), a physical mixture of lipopeptide 24 showed slow tumour growth. It is particularly
lipid 1 conjugated with 8Qmin (27) and lipid 1 conjugated with E643-57 noteworthy that 46% of mice (6 out of 13 mice) treated with
(
28) (100 μg/100 μL sterile PBS, 1:1) (Scheme 5), 8Qmin/E643-57 lipopeptide 24 were tumour free after 60 days (Figure 6c). The
epitopes conjugated with Pam2Cys (29) (100 μg/100 μL sterile PBS) therapeutic efficacy of 24 was similar to that of positive control (a
as a positive control, or PBS (100 μL) as a negative control. The mixture of 8Qmin + E643-57 + ISA51) (Figures 5b and 6b-d). We also
Kaplan-Meier survival curve (Figure 5a) showed that all of the mice demonstrated that both E744-57 and E643-57 peptides were active in
treated with PBS were euthanised due to tumour burden by day 45. lipopeptide 24 by assessing recall IFN-γ production by CD8 T-cells
In contrast, mice treated with lipopeptide 24 and 25 demonstrated from immunised mice in response to MHC class I-restricted E7
3
8% (3 out of 8 mice) and 25% (2 out of 8 mice) survival rates, (RAHYNIVTF) or E6 (YDFAFRDL) peptide restimulation by ELISPOT
respectively, which was significantly better than that for mice (Fig. 7).
treated with the positive control (8Qmin/E643-57-Pam2Cys (29), 0%
survival, 0 out of 8 mice). Among tested groups, the slowest tumour
growth was observed in mice immunised with vaccine candidate 24 Conclusions
(
Figure 6a). The physical mixture of 27 and 28 did not slow down
tumour growth significantly and only one mouse treated with the
mixture survived to the end of the experiment. We proposed that We established a synthetic double conjugation pathway to
the physical mixture of 27 and 28 would allow each epitope to be develop multiantigenic lipopeptide conjugates as self-
taken up by different APCs, thus the immune stimulating effect of T- adjuvanting therapeutic vaccine candidates to treat HPV-
helper epitope present in 8Qmin may not enhance the immune related cancers. The method involved a Michael addition
response against the E643-57 epitope. It was reported that the co- mercapto-acryloyl reaction between two unprotected peptides
recognition of T-helper and CTL epitopes by the same APC was followed by an azide-alkyne click reaction to give the final
2
2
essential for the efficient stimulation of cellular immunity.
Interestingly, the biological study showed that Pam2Cys analogue moieties were synthesised, conjugated with the multiantigenic
29) and the most hydrophobic vaccine candidate 26 induced very branched peptide and were found to stimulate significantly
lipopeptide products. Three novel lipidic self-adjuvanting
(
weak antitumour responses. In tumour challenge, 0/5 and 1/5 mice better survival in an in vivo murine HPV model than mice
survived on day 60 for 29 and 26, respectively (Figure 5a) despite treated with the Pam2Cys analogue 29, without an external
the well-proven ability of Pam2Cys to induce cellular immune adjuvant and after only a single immunization.
2
3
responses. This might be explained by the formation of large
Our double conjugation strategy provided an overall yield 50-
aggregates (>5 µm in diameter) by conjugates 26 and 29 while 80%; can be applied to a wide variety of synthetic applications;
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was simple to perform; and can be applied on unprotected backscatter system. Multiplicate measurements were performed at
peptides. This strategy could also be used to rapidly produce 25 °C with scattering angle of 173° using disposable cuvettes and
libraries of different vaccine constructs from a small selection the number-average hydrodynamic particle diameters are reported.
of starting components (e.g. a variety of epitopes and Synthesis of 10-Butoxydecan-1-ol (6)
26
adjuvants). It is anticipated that this technique will be widely Alcohol 6 was synthesised following a reported procedure.
used in the chemical synthesis of branched multiantigenic Sodium hydride (60% dispersion in oil, 400 mg, 10.00 mmol, 2
peptides and self-adjuvanting vaccines.
equiv.) was added to a solution of 1,10-decanediol (4) (1.74 g, 10.00
mmol, 2 equiv.) in dry DMF (30 mL) and the mixture was stirred for
1
0
min under
a
nitrogen atmosphere.
A
mixture of
Experimental Section
tetrabutylammonium iodide (TBAI) (93 mg, 0.25 mmol, 0.05 equiv.)
and butyl bromide (537 µL, 0.69 g, 5 mmol, 1 equiv.) was added and
the mixture was sonicated for 3 h. The reaction mixture was
evaporated in vacuo and the residue was taken up with a mixture of
Materials. 1,3-di(hydroxymethyl)-5-(prop-2-ynyloxy)benzene was
24
prepared as reported. Protected
from Novabiochem (Läufelfingen, Switzerland) and Mimotopes
Melbourne, Australia). pMBHA resin was purchased from Peptides
L-amino acids were purchased
(
5
% HCl (100 mL) and ethyl acetate (EtOAc) (100 mL). The aqueous
layer was further extracted with EtOAc (2 x 100 mL). The combined
organic layers were washed with 0.2 M Na (1 x 50 mL), water
1 x 50 mL), dried over anhydrous MgSO , filtered and evaporated
International (Kentucky, USA). Rink amide MBHA resin, N,N’-
dimethylformamide (DMF), dichloromethane (DCM), methanol,
N,N’-diisopropylethylamine (DIPEA), piperidine and trifluoroacetic
acid (TFA) were obtained from Merck (Darmstadt, Germany).
Copper wire was purchased from Aldrich (Castle Hill, Australia).
2 2 3
S O
(
4
in vacuo to afford the crude product as yellow oil. The crude
product was purified by silica flash column chromatography (a
gradient elution of 0 – 10% EtOAc in hexane) to afford the
(Dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3-
yloxy)-methanim-inium hexafluorophosphate (HATU) was
butoxydecanol (6) as a colourless oil (487 mg, 42%), R
EtOAc in hexane, KMnO dip). H NMR (500 MHz, CDCl
4 3
f
: 0.20 (10%
) δ 3.59 (t, J
.7 Hz, 2H), 3.37 (t, J 6.7 Hz, 2H), 3.35 (t, J 6.7 Hz, 2H), 1.58 (s, OH,
H), 1.55 – 1.49 (m, 6H), 1.37 – 1.22 (m, 14H), 0.88 (t, J 7.4 Hz, 3H).
1
purchased from Mimotopes (Melbourne, Australia). HPLC grade
acetonitrile was obtained from Labscan (Bangkok, Thailand). All
other reagents were obtained at the highest available purity from
Sigma-Aldrich (Castle Hill, NSW, Australia). Anhydrous hydrofluoric
acid (HF) was supplied by BOC gases (Sydney, NSW, Australia). A
Kel-F HF apparatus (Peptide Institute, Osaka, Japan) was used for HF
6
1
1
3
3
C NMR (125 MHz, CDCl ) δ 70.9, 70.6, 63.0, 32.8, 31.8, 29.7, 29.49,
2
9.48, 29.43, 29.37, 26.1, 25.7, 19.3, 13.9; ESI-MS, m/z: 231 [M +
+
+
H] . HRMS calculated for C14
Synthesis of 6-(octyloxy)hexan-1-ol (7)
Alcohol 7 was synthesised following a reported procedure.
2
H30NaO 253.2138, found 253.2130.
cleavage. ESI-MS was performed using
a Perkin-Elmer-Sciex
26
API3000 instrument with Analyst 1.4 software (Applied
Biosystems/MDS Sciex, Toronto, Canada). High-resolution
electrospray ionization mass spectra measurements were obtained
on a Bruker micrOTOF mass spectrometer by direct infusion in
MeCN at 3 μL/min using sodium formate clusters as an internal
calibrant. Nuclear magnetic resonance (NMR) spectra were
recorded with a Bruker Avance 300, 500 or 600 MHz spectrometer
Sodium hydride (60% dispersion in oil, 400 mg, 10.0 mmol, 2 equiv.)
was added to a solution of diol 5 (1.2 g, 10.00 mmol, 2 equiv.) in dry
DMF (30 mL) and the mixture was stirred for 10 min under a
nitrogen atmosphere. A mixture of TBAI (93 mg, 0.25 mmol, 0.05
equiv.) and n-octyl bromide (864 μL, 0.97 g, 5 mmol, 1 equiv.) was
added and the mixture was sonicated for 2 h. The reaction mixture
was evaporated in vacuo and the residue was taken up with a
mixture of 5% HCl (100 mL) and ethyl acetate (EtOAc) (100 mL). The
aqueous layer was further extracted with EtOAc (2 x 100 mL). The
(
Bruker Biospin, Germany). Chemical shifts are reported in parts per
million (ppm) on a δ scale, relative to the solvent peak (CDCl
.24, δ 77.0). Coupling constants (J) are reported in hertz (Hz) and
3
δ
H
7
C
peak multiplicities described as singlet (s), doublet (d), doublet of
doublet (dd), triplet (t), quartet (q), multiplet (m), or broad (br).
Analytical RP-HPLC was performed using Shimadzu (Kyoto, Japan)
instrumentation (DGU-20A5, LC-20AB, SIL-20ACHT, SPD-M10AVP)
with a 1 mL min flow rate and detection at 214 nm and/or
evaporative light scattering detector (ELSD). Separation was
achieved using a 0-100% linear gradient of solvent B over 40 min
combined organic layers were washed with 0.2 M Na
mL), water (1 x 50 mL), dried over anhydrous MgSO
evaporated in vacuo to afford the crude product. The crude product
was purified by silica flash column chromatography (a gradient
elution of 0 – 10% EtOAc in hexane) to afford the alcohol 7 as a
2
S
2
O
3
(1 x 50
, filtered and
4
−1
colourless oil (504 mg, 44%), R
f
: 0.20 (10% EtOAc in hexane, KMnO
) δ 3.60 (t, J 6.6 Hz, 2H), 3.37 (t, J 6.6
Hz, 2H), 3.36 (t, J 6.8 Hz, 2H), 1.58 – 1.50 (m, 6H), 1.34 (quintet, J
4
1
dip). H NMR (500 MHz, CDCl
3
with Method A (0.1% TFA/H O as solvent A and 90% MeCN/0.1%
2
1
3
2 2
TFA/H O as solvent B) and/or Method B (0.1% TFA/H O as solvent
3
.7 Hz, 4H), 1.29 – 1.24 (m, 10H), 0.84 (t, J 7.0 Hz, 3H). C NMR (125
A and 90% MeOH/0.1% TFA/H O as solvent B) on either a Vydac
2
MHz, CDCl ) δ 71.0, 70.8, 62.8, 32.7, 31.8, 29.71, 29.68, 29.4, 29.2,
3
+
analytical C4 column (214TP54; 5 ꢀm, 4.6 mm x 250 mm) or a Vydac
analytical C18 column (218TP54; 5 ꢀm, 4.6 mm x 250 mm). calculated for C H NaO 253.2138, found 253.2138.
2
6.2, 26.0, 25.6, 22.6, 14.1; ESI-MS, m/z: 253 [M + Na] . HRMS
+
14
30
2
Preparative RP-HPLC was performed on Shimadzu (Kyoto, Japan) Synthesis of 5,16,20,32-tetraoxahexatriacontan-18-ol (9)
instrumentation (either LC-20AT, SIL-10A, CBM-20A, SPD-20AV, Alcohol 9 was synthesised following a reported procedure. A mix
FRC-10A or LC-20AP x 2, CBM-20A, SPD-20A, FRC-10A) in linear of butoxydecanol (6) (165 mg, 0.72 mmol,
equiv.),
2
6
2
gradient mode using a 5-20 mL/min flow rate, with detection at 230 Tetrabutylammonium bromide (TBABr) (12 mg, 0.036 mmol, 0.1
nm. Separations were performed with solvent A and solvent B on a equiv.) and NaOH (32 mg, 0.79 mmol, 2.2 equiv.) was stirred for 10
Vydac preparative C18 column (218TP1022; 10 ꢀm, 22 mm x 250 min at room temperature. Epichlorohydrin (28 µL, 33 mg, 0.36
°
mm), Vydac semi-preparative C18 column (218TP510; 5 ꢀm, 10 mm mmol, 1 equiv.) was added and the mixture was stirred at 30 C for
x 250 mm) or Vydac semi-preparative C4 column (214TP510; 5 ꢀm, 14 h and then sonicated for 5 h. The reaction mixture was taken up
1
0 mm x 250 mm). Flash chromatography was performed on Merck with a mixture of 5% HCl (75 mL) and Et
2
O (50 mL). The aqueous
O (2 x 50 mL). The combined
dynamic light scattering (DLS) using a Malvern Zetasizer Nano Series organic layers were washed with water (1 x 50 mL), dried over
2
5
Kieselgel 60 as described by Still. Particle size was measured by layer was further extracted with Et
2
with DTS software. Sizes were analysed using a non-invasive anhydrous MgSO
, filtered and evaporated in vacuo to afford the
J. Name., 2013, 00, 1-3 | 7
4
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Chemical Science
Page 8 of 13
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DOI: 10.1039/C5SC03859F
ARTICLE
Journal Name
crude product of diglyceride alcohol 9 as a yellow oil. The crude 2.38 (t, J 2.4 Hz, 1H), 1.56 – 1.50 (m, 12H), 1.38 – 1.23 (m, 28H),
1
3
product was taken on to the next reaction without further 0.90 (t, J 7.5 Hz, 6H). C NMR (150 MHz, CDCl
3
) δ 80.3, 76.6, 74.0,
purification or characterization. R : 0.77 (20% EtOAc in DCM, 71.7, 71.0, 70.8, 70.6, 57.6, 31.9, 29.8, 29.68, 29.62, 29.54, 29.49,
f
+
Ce(SO
4
)
2
dip).
29.45, 26.2, 26.1, 19.4, 13.9; ESI-MS, m/z: 577 [M + Na] . HRMS
+
Synthesis of 9,16,20,27-tetraoxapentatriacontan-18-ol (10)
calculated for C H NaO 577.4802, found 577.4804.
34
66
5
A mix of alcohol 7 (230 mg, 1.00 mmol, 3 equiv.), TBABr (11 mg, Synthesis of 18-(prop-2-yn-1-yloxy)-9,16,20,27-tetraoxapentatria
.03 mmol, 0.1 equiv.) and NaOH (44 mg, 1.10 mmol, 3.3 equiv.) contane or lipoalkyne (2)
0
was stirred for 10 min. Epichlorohydrin (26 µL, 31 mg, 0.33 mmol, 1 A solution of alcohol 10 (36 mg, 0.10 mmol, 1 equiv.) in dry THF (2
equiv.) was added and the mixture was stirred at 30 °C for 14 h and mL) was added dropwise to a suspension of NaH (60% dispersion in
then sonicated for 5 h. The reaction mixture was taken up with a oil, 12 mg, 0.30 mmol, 3 equiv.) in dry THF (1 mL) over a period of 5
o
mixture of 5% HCl (75 mL) and Et O (50 mL). The aqueous layer was min at 0 C under a nitrogen atmosphere. The reaction mixture was
2
o
further extracted with Et
2
O (2 x 50 mL). The combined organic stirred for 15 min at 0 C. A solution of propargyl bromide (80% in
layers were washed with water (1 x 50 mL), dried over anhydrous toluene, 33 µL, 45 mg, 0.30 mmol, 3 equiv.) in dry THF (1 mL) was
o
MgSO
product. The crude product was purified by silica flash column stirred for 14 h. The solvent was evaporated in vacuo and the
chromatography (a gradient elution of 0 – 20% EtOAc in DCM) to residue was taken up with a mixture of water (50 mL) and Et O (100
afford the branched alcohol 10 (74 mg, 43%), R : 0.50 (15% EtOAc in mL). The aqueous layer was further extracted with Et O (100
) δ 3.93 – 3.87 (m, mL).The combined organic layers were dried over anhydrous
H), 3.46 – 3.37 (m, 8H), 3.354 (t, J 6.6 Hz, 4H), 3.349 (t, J 6.8 Hz, MgSO , filtered and evaporated in vacuo to afford the crude
H), 2.29 (br s, 1H), 1.57 - 1.49 (m, 12H), 1.32 (quintet, J 3.7 Hz, 8H), product.The crude product was purified by silica flash column
4
, filtered and evaporated in vacuo to afford the crude added to the reaction mixture at 0 C over 2 min. The mixture was
2
f
2
1
4 2 3
DCM, Ce(SO ) dip). H NMR (400 MHz, CDCl
1
4
1
4
1
3
.28 – 1.24 (m, 20H), 0.84 (t, J 6.9 Hz, 6H). C NMR (100 MHz, chromatography (a gradient elution of 0 – 20% EtOAc in hexane) to
) δ 71.8, 71.5, 71.0, 70.8, 69.4, 31.8, 29.72, 29.68, 29.5, 29.4, afford the alkyne derivative 2 (18 mg, 47%), R : 0.12 (5% EtOAc in
) δ 4.31 (d, J 2.4 Hz,
CDCl
4 2 3
9.2, 26.2, 26.0, 25.9, 22.6, 14.0; ESI-MS, m/z: 539 [M + Na] . HRMS hexane, Ce(SO ) dip). H NMR (400 MHz, CDCl
3
f
+
1
2
+
calculated for C31
Synthesis of 1,3-bis(hexadecyloxy)propan-2-ol (11)
H64NaO
5
539.4646, found 539.4648.
2H), 3.82 (quintet, J 5.2 Hz, 1H), 3.53 – 3.46 (m, 4H), 3.44 – 3.39 (m,
4H), 3.363 (t, J 6.7 Hz, 4H), 3.359 (t, J 6.7 Hz, 4H), 2.38 (t, J 2.4 Hz,
A mix of hexadecane-1-ol (8, 485 mg, 2.00 mmol, 3 equiv.), TBABr 1H), 1.56 – 1.50 (m, 12H), 1.33 (quintet, J 3.7 Hz, 8H), 1.29 – 1.25
1
3
(
22 mg, 0.07 mmol, 0.1 equiv.) and NaOH (88 mg, 2.20 mmol, 3.3 (m, 20H), 0.86 (t, J 6.9 Hz, 6H). C NMR (100 MHz, CDCl
3
) δ 80.3,
equiv.) was stirred for 10 min. Epichlorohydrin (53 µL, 62 mg, 0.67 76.6, 74.0, 71.6, 71.0, 70.9, 70.8, 57.6, 31.8, 29.8, 29.7, 29.6, 29.5,
O
+
mmol, 1 equiv.) was added and the mixture was stirred at 60 C for 29.3, 26.2, 26.1, 26.0, 22.6, 14.1; ESI-MS, m/z: 577 [M + Na] . HRMS
+
1
4 h and then sonicated for 5 h. The reaction mixture was taken up calculated for C34
with a mixture of 5% HCl (75 mL) and Et O (50 mL). The aqueous Synthesis of 1-(3-(hexadecyloxy)-2-(prop-2-yn-1-yloxy) propoxy)
layer was further extracted with Et O (2 x 50 mL). The combined hexadecane or lipoalkyne (3)
organic layers were washed with water (1 x 50 mL), dried over The branched alcohol 11 (51 mg, 0.10 mmol, 1 equiv.) in dry THF (2
anhydrous MgSO , filtered and evaporated in vacuo to afford the mL) was added dropwise to a suspension of NaH (60% dispersion in
5
H66NaO 577.4802, found 577.4809.
2
2
4
crude product. The crude product was purified by silica flash oil, 12 mg, 0.30 mmol, 3 equiv.) in dry THF (1 mL) over a period of 5
O
column chromatography (a gradient elution of 0 – 30% EtOAc in min at 0 C under a nitrogen atmosphere. The reaction mixture was
O
hexane) to afford the branched alcohol 11 (302 mg, 83%), R
f
: 0.34 stirred for 15 min at 0 C. A solution of propargyl bromide (80% in
1
(10% EtOAc in hexane, Ce(SO
4
)
2
dip). H NMR (400 MHz, CDCl
3
) δ toluene, 33 µL, 45 mg, 0.30 mmol, 3 equiv.) in dry THF (1 mL) was
.95 – 3.89 (m, 1H), 3.62 (br s, 1H), 3.48 – 3.39 (m, 7H), 2.46 (d, J 4.2 added to the reaction mixture at 0 C over 2 min. The mixture was
O
3
Hz, 1H), 1.55 (quintet, J 7.0 Hz, 4H), 1.29 – 1.20 (m, 52H), 0.86 (t, J stirred for 14 h. The solvent was evaporated in vacuo and the
1
3
6
2
.9 Hz, 6H). C NMR (100 MHz, CDCl
3
) δ 71.8, 71.7, 69.5, 31.9, 29.7, residue was taken up with a mixture of water (50 mL) and Et
2
O (100
O (100
mL).The combined organic layers were dried over anhydrous
MgSO , filtered and evaporated in vacuo to afford the crude
product. The crude product was purified by silica flash column
+
9.6, 29.5, 29.4, 26.1, 22.7, 14.1; ESI-MS, m/z: 564 [M + Na] . HRMS mL). The aqueous layer was further extracted with Et
2
+
3
calculated for C35H72NaO 563.5374, found 563.5373.
Synthesis of 18-(prop-2-yn-1-yloxy)-5,16,20,31-tetraoxapentatria
contane or lipoalkyne (1)
4
Sodium hydride (60% dispersion in oil, 16 mg, 0.39 mmol, 1.1 chromatography (a gradient elution of 0 – 10% EtOAc in hexane) to
equiv.) was added to a solution of the crude diglyceride alcohol 9 afford the alkyne derivative 3 (31 mg, 56%), R : 0.36 (5% EtOAc in
dip). H NMR (400 MHz, CDCl ) δ 4.31 (d, J 2.32
f
1
(184 mg, 0.36 mmol, 1 equiv.) in dry DMF (5 mL) and the mixture hexane, Ce(SO
4
)
2
3
was stirred for 10 min under a nitrogen atmosphere. Propargyl Hz, 2H), 3.83 (quintet, J 5.2 Hz, 1H), 3.54 – 3.47 (m, 4H), 3.45 – 3.39
bromide (80% in toluene, 116 µL, 161 mg, 1.08 mmol, 3 equiv.) was (m, 4H), 2.37 (t, J 2.3 Hz, 1H), 1.54 (quintet, J 6.9 Hz, 4H), 1.25 –
1
3
3
added and the mixture was sonicated for 1 h and stirred overnight. 1.20 (m, 52H), 0.86 (t, J 6.8 Hz, 6H). C NMR (100 MHz, CDCl ) δ
Afterwards, the solvent was evaporated in vacuo and the residue 80.3, 76.6, 74.0, 71.7, 70.8, 57.6, 31.9, 29.69, 29.65, 29.6, 29.5,
+
was taken up with a mixture of water (50 mL) and EtOAc (50 mL). 29.4, 26.1, 22.7, 14.1; ESI-MS, m/z: 602 [M + Na] . HRMS calculated
+
The organic layer was dried over anhydrous MgSO
4
, filtered and for C38
H74NaO
3
601.5530, found 601.5535.
evaporated in vacuo to afford the crude product as yellow oil. The Synthesis of 8Qmin Peptide.
crude product was purified by silica flash column chromatography 8Qmin epitope (QAEPDRAHYNIVTF; E744-57) was synthesised by
(
(
a gradient elution of 0 -10% EtOAc in DCM) to afford the lipoalkyne manual stepwise SPPS on pMBHA resin (substitution ratio: 0.45
1) as a yellow oil (57 mg, 29%) over two steps, R : 0.29 (1% EtOAc in mmol/g, 0.2 mmol scale, 0.44g) using HATU/DIPEA Boc-chemistry.
) δ 4.32 (d, J 2.4 Hz, Boc-amino acids were preactivated for 1 min prior to their addition
H), 3.83 (quintet, J 5.1 Hz, 1H), 3.51 (dd, J 10.4 Hz, J 17.0 Hz, 2H), to the resin. The activation of amino acids was achieved by
.50 (dd, J 10.4 Hz, J 17.9 Hz, 2H), 3.411 (dd, J 6.6 Hz, J 11.9 Hz, 2H), dissolving Boc-amino acid (0.84 mmol, 4.2 equiv.), in 0.5 M
.411 (dd, J 7.7 Hz, J 16.2 Hz, 2H), 3.37 (dd, J 6.7 Hz, J 13.3 Hz, 8H), HATU/DMF solution (1.6 mL, 0.8 mmol, 4.0 equiv.) followed by the
f
1
4 2 3
DCM, Ce(SO ) dip). H NMR (600 MHz, CDCl
2
3
3
8
| J. Name., 2012, 00, 1-3
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Page 9 of 13
Journal Name
Plea sC eh de om ni oc ta al dS j uc si et nm c ae rgins
View Article Online
DOI: 10.1039/C5SC03859F
ARTICLE
addition of DIPEA (0.22 mL, 1.24 mmol, 6.2 equiv.). Coupling cycle deprotection of Thr, Val, and Ile were performed with 2% of 1,8-
consisted of a Boc deprotection step with neat TFA (2 × 1 min, at rt), diazabicycloundec-7-ene (DBU) in DMF (twice, 5 and 10 min)
a 1 min DMF flow-wash, followed by coupling with 4.2 equiv. of instead of 20% piperidine in DMF. The attachment of azidoacetic
preactivated Boc-amino acids (2 × 1 h). For peptides containing acid (4.2 equiv.) was achieved using HATU (3 equiv.)/DIPEA (4.2
His(DNP) residues, the DNP (2,4-dinitrophenyl) group was cleaved equiv.) at room temperature (2 × 1 h) and the reaction mixture was
by treating the resin with 20% (v/v) β-mercaptoethanol and 10% covered and protected from light with aluminum foil. The crude
(
v/v) DIPEA in DMF for 2 × 1 h treatments prior to peptide cleavage. product was purified by a preparative RP-HPLC on C-18 column with
Upon completion of synthesis and removal of the dinitrophenyl a 20-40% solvent B gradient over 20 min. HPLC analysis (C-18
DNP) protecting group, the resin was washed with DMF, DCM, and column, Method A): t = 18.3 min, purity > 95%. Yield: 72%. ESI-MS:
(
R
2
+
MeOH, then dried (vacuum desiccator). The peptide was cleaved m/z 924.0 (calc 924.0) [M+2H] ; MW 1846.
from the resin using HF, with p-cresol as a scavenger. The cleaved Synthesis of N-terminal acryloyl E643-57 (22).
peptide was precipitated, filtered, and washed thoroughly with ice- N-terminal acryloyl E643-57 peptide epitope (CH
2
=CHCO-
2 2
cold Et O and dissolved in 50% MeCN/0.1% TFA/H
O. After QLLRREVYDFAFRDL) was synthesised following the general manual
lyophilization, the crude peptide was obtained as an amorphous stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. The
powder. The product was purified by a preparative RP-HPLC on C18 coupling of acrylic acid (4.2 equiv.) was achieved using HATU (4
column with a 15-35% solvent B gradient over 20 min. HPLC analysis equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h). The crude
(
C18 column, Method A): t
= 16.7 min, purity > 95%. Yield: 27%. product was purified by a preparative RP-HPLC on C-18 column with
R
ESI-MS: m/z 1661.1 (calc 1660.8) [M+H] ; 830.8 (calc 830.9) a 35-55% solvent B gradient over 20 min. HPLC analysis (C-18
+
2
+
[
M+2H] ; MW 1659.8.
General procedure of manual stepwise SPPS on rink amide MBHA m/z 998.2 (calc 998.1) [M+2H] ; 665.8 (calc 665.8) [M+3H] ; MW
resin - Fmoc-chemistry
1994.3.
R
column, Method A): t = 22.7 min, purity > 95%. Yield: 33%. ESI-MS:
2
+
3+
Peptides were synthesised by manual stepwise SPPS on rink amide Preparation of Guanidine Buffer
MBHA resin (substitution ratio: 0.60 mmol/g, 0.2 mmol scale, 0.33 6 M guanidine, 50 mM sodium phosphate, 20% acetonitrile, 5 mM
g) using HATU/DIPEA Fmoc-chemistry. Amino acid activation was EDTA, ~pH 7.3.
achieved by dissolving Fmoc-amino acid (0.84 mmol, 4.2 equiv.), in Synthesis of multiantigenic peptide azide (23) through mercapto-
0
.5 M HATU/DMF solution (1.6 mL, 0.8 mmol, 4.0 equiv.) followed acryloyl conjugation
by the addition of DIPEA (146 ꢀL, 0.84 mmol, 4.2 equiv.). Coupling A mixture of the two peptide epitopes acryloyl E643-57 (22) (7.2 mg,
cycle consisted of Fmoc deprotection with 20% of piperidine in DMF 3 µmol, 1.0 equiv.) and 8Qmin mercapto-azide (21) (13.4 mg, 6 µmol,
(
twice, 10 and 20 min), a 1 min DMF flow-wash, followed by 2 equiv.) was dissolved in a guanidine buffer at ~pH 7.3. The
coupling with 4.2 equiv. of preactivated Fmoc-amino acids (2 × 1 h). reaction mixture was incubated at 37 °C for 48 h. The progress of
Upon completion of synthesis, the resin was washed with DMF, the reaction was monitored by analytical HPLC until the acryloyl
DCM, and MeOH, then dried (vacuum desiccator). The cleavage of E643-57 (22) was completely consumed. The reaction mixture was
model mercapto-azide was carried out by stirring the resin in the purified using a semi-preparative HPLC on a C-18 column (20-60%
solution of TFA (99%)/triisopropylsilane/water (95:2.5:2.5) for 4 h. solvent B over 60 min). After lyophilization, the pure azide
The cleaved peptide was precipitated, filtered, and washed with ice- derivative 23 was obtained as an amorphous white powder. The
cold Et
amorphous powder.
Synthesis of E643-57
E643-57 epitope (QLLRREVYDFAFRDL; E643-57
2
O. After lyophilization, the crude peptide was obtained as an product was detected using analytical HPLC analysis (C-4 column,
Method A), t = 21.8 min, purity > 97% and (C18 column, Method
A), t = 21.4 min, purity > 95%. Yield: (12.2 mg, 90%). ESI-MS: m/z
was synthesised 1921.5 (calc 1921.1) [M+2H] ; 1281.3 (calc 1281.1) [M+3H] ; 961.2
R
.
R
2
+
3+
)
4
+
5+
following the general manual stepwise SPPS HATU/DIPEA Fmoc- (calc 961.1) [M+4H] ; 768.9 (calc 769.1) [M+5H] ; MW 3840.3.
chemistry procedure. The crude product was purified by a Synthesis of vaccine candidate lipopeptide 24
-
4
preparative RP-HPLC on C-18 column with 25-45% solvent B A mixture of azide derivative 23 (3.3 mg, 7.5 x 10 mmol, 1 equiv.)
-
4
gradient over 20 min. HPLC analysis (C-18 column, Method A): t
R
=
and the lipoalkyne 1 (0.6 mg, 11.3 x 10 mmol, 1.5 equiv.) was
1
[
9.8 min, purity > 95%. Yield: 84%. ESI-MS: m/z 970.9 (calc 971.1) dissolved in DMF (1 mL), and copper wire (80 mg) was added. The
2
+
3+
M+2H] ; 647.8 (calc 647.7) [M+3H] ; MW 1940.2.
Synthesis of Azidoacetic Acid (N CH CO
synthesised using a similar method to the published procedure.
Sodium azide (6.0 g, 92.3 mmol, 3.0 equiv.) was dissolved in H
O (10 the reaction was monitored by analytical HPLC (C-4 column) and
air in the reaction mixture was removed by nitrogen bubbling. The
H). Azidoacetic acid was reaction mixture was covered and protected from light with
3
2
2
27
aluminum foil and stirred at 50 °C under nitrogen. The progress of
2
mL) and bromoacetic acid (4.3 g, 30.8 mmol, 1.0 equiv.) was added. ESI-MS until the peptide 23 was completely consumed after 4 h.
The reaction mixture was covered and protected from light with The reaction mixture was purified using a semi-preparative HPLC on
aluminum foil. The reaction was stirred continuously in an ice bath a C-4 column (35-75% solvent B over 60 min). After lyophilization,
for 24 h and subsequently acidified with 32% HCl (10 mL). The the pure lipopeptide 24 was obtained as an amorphous white
product was then extracted with Et
anhydrous MgSO
and the solvent was evaporated under vacuum. A) t
2
O (4 x 50 mL), dried over powder. Compound 24 was analysed by HPLC (C-4 column, Method
R
= 29.9 min, purity > 97% (detected by UV at 214 nm) and t =
4
R
The final product was obtained as a colorless oil (2.95 g, 95%) after 30.0 min, purity > 96% (detected by evaporative light scattering
prolonged evaporation under vacuum to remove organic solvent detector). Yield: (3.2 mg, 87%).
1
3+
and the last traces of water. H NMR (300 MHz, CDCl
3
) δ 3.98 (s, ESI-MS: m/z 1466.2 (calc 1466.1) [M+3H] ; 1100.0 (calc 1099.8)
) δ 173.7, 50.0.
1
3
4+
5+
2
H), 10.60 (br s, OH, 1H). C NMR (100 MHz, CDCl
Synthesis of N-terminus 8Qmin mercapto-azide (21).
3
[M+4H] ; 880.2 (calc 880.0) [M+5H] ; MW 4395.1.
Synthesis of vaccine candidate lipopeptide 25
N-terminus 8Qmin mercapto-azide peptide epitope (N
3
CH
2
CO- A mixture of azide derivative 23 (3.0 mg, 0.7 µmol, 1 equiv.) and the
CQAEPDRAHYNIVTF) was synthesised following the general manual lipoalkyne 2 (3.0 mg, 1.8 µmol, 2.5 equiv.) was dissolved in DMF (1
stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. Fmoc mL), and copper wire (60 mg) was added. The air in the reaction
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mixture was removed by nitrogen bubbling. The reaction mixture mixture was covered and protected from light with aluminum foil
was covered and protected from light with aluminum foil and and stirred at 50 °C under nitrogen. The progress of the reaction
stirred at 50 °C under nitrogen. The progress of the reaction was was monitored by analytical HPLC (C-4 column) and ESI-MS until the
monitored by analytical HPLC (C-4 column) and ESI-MS until the 8Qmin-azide was completely consumed after 3 h. The reaction
peptide 23 was completely consumed after 3 h. The reaction mixture was purified using a semi-preparative HPLC on a C-4
mixture was purified using a semi-preparative HPLC on a C-4 column (35-75% solvent B over 60 min). After lyophilization, the
column (40-80% solvent B over 60 min). After lyophilization, the pure product 27 was obtained as an amorphous white powder.
pure lipopeptide 25 was obtained as an amorphous white powder. Compound 27 was analysed by HPLC (C-4 column, Method A) t
R
=
Compound 25 was analysed by HPLC (C-4 column, Method A) t = 30.9 min, purity > 97% (detected by UV at 214 nm) and t = 30.4
R
R
2
9.9 min and t
R
= 36.2 min (C-4 column, Method B), purity > 97% min, purity > 96% (detected by evaporative light scattering
detector). Yield: (1.7 mg, 32%). ESI-MS: m/z 1150.1 (calc 1149.9)
(detected by UV at 214 nm). Yield: (2.7 mg, 80%).
3
+
2+
3+
ESI-MS: m/z 1466.2 (calc 1466.1) [M+3H] ; 1100.0 (calc 1099.8) [M+2H] ; 767.2 (calc 766.9) [M+3H] ; MW 2297.7
4
+
5+
[M+4H] ; 880.0 (calc 880.0) [M+5H] ; MW 4395.1.
Synthesis of lipid 1 conjugated with E64 (28)
3-57
A mixture of E643-57-azide (5.0 mg, 2 x 10 mmol, 1 equiv.) and the
-3
Synthesis of vaccine candidate lipopeptide 26
-3
A mixture of azide derivative 23 (3.8 mg, 0.9 µmol, 1 equiv.) and the lipoalkyne 1 (1.7 mg, 3 x 10 mmol, 1.5 equiv.) was dissolved in
lipoalkyne 3 (2.6 mg, 4.5 µmol, 5 equiv.) was dissolved in a mixture DMF (1 mL), and copper wire (80 mg) was added. The air in the
of DMF (0.8 mL) and DMSO (0.5 mL), and copper wire (80 mg) was reaction mixture was removed by nitrogen bubbling. The reaction
added. The air in the reaction mixture was removed by nitrogen mixture was covered and protected from light with aluminum foil
bubbling. The reaction mixture was covered and protected from and stirred at 50 °C under nitrogen. The progress of the reaction
light with aluminum foil and stirred at 50 °C under nitrogen. The was monitored by analytical HPLC (C-4 column) and ESI-MS until the
progress of the reaction was monitored by analytical HPLC (C-4 E643-57-azide was completely consumed after 6 h. The reaction
column) and ESI-MS until the peptide 23 was completely consumed mixture was purified using a semi-preparative HPLC on a C-4
after 7 h. The reaction mixture was purified using a semi- column (35-75% solvent B over 60 min). After lyophilization, the
preparative HPLC on a C-4 column (45-85% solvent B over 60 min). pure product 28 was obtained as an amorphous white powder.
After lyophilization, the pure lipopeptide 26 was obtained as an Compound 28 was analysed by HPLC (C-4 column, Method A) t
R
=
amorphous white powder. Compound 26 was analysed by HPLC (C- 27.4 min, purity > 97% (detected by UV at 214 nm). Yield: (3.6 mg,
2
+
4
column, Method A) t
R
= 35.3 min, purity > 97% (detected by UV at 58%). ESI-MS: m/z 1289.8 (calc 1290.1) [M+2H] ; 860.1 (calc 860.4)
3
+
214 nm). Yield: (2.1 mg, 49%). ESI-MS: m/z 1474.2 (calc 1474.1) [M+3H] ; MW 2578.1
3
+
4+
28
[
M+3H] ; 1106.0 (calc 1105.8) [M+4H] ; 885.0 (calc 884.9) Synthesis of S-(2,3-dihydroxypropyl) Cysteine (Dhc-OH)
5
+
[M+5H] ; MW 4419.2.
As shown in scheme 6, a mixture of
L
-cysteine hydrochloride (1 g, 6
Synthesis of N-terminus 8Qmin-azide.
N-terminus 8Qmin-azide peptide
mmol), 3-bromo-propan-1,2-diol (1.4 g, 0.79 mL, 9 mmol) and
3 2
(N CH CO- triethylamine (2 g, 2.7 mL, 19 mmol) in water (5 mL) was covered
epitope
QAEPDRAHYNIVTF) was synthesised following the general manual and protected from light with aluminum foil and kept for 3 days.
stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. Fmoc The reaction mixture was evaporated in vacuo and the residue was
deprotection of Thr, Val, and Ile were performed with 2% of 1,8- washed with acetone (3 x 15 mL) and dried to give Dhc-OH as a
diazabicycloundec-7-ene (DBU) in DMF (twice, 5 and 10 min) white solid (0.8 g, 3.5 mmol, 67%).
instead of 20% piperidine in DMF. The attachment of azidoacetic Synthesis of N-fluorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)
2
8
acid (4.2 equiv.) was achieved using HATU (3 equiv.)/DIPEA (4.2 cysteine (Fmoc-Dhc-OH)
equiv.) at room temperature (2 × 1 h) and the reaction mixture was As shown in scheme 6, a solution of fluorenylmethoxycarbonyl-N-
covered and protected from light with aluminum foil. The crude hydroxysuccinimide (1.15 g, 3.5 mmol) in acetonitrile (10 mL) was
product was purified by a preparative RP-HPLC on C-18 column with added to a solution of Dhc-OH (0.8 g, 3.5 mmol) in 9% sodium
a 15-35% solvent B gradient over 20 min. HPLC analysis (C-18 carbonate (10 mL). The reaction mixture was stirred at room
column, Method A): t
R
= 17.9 min, purity > 95%. Yield: 80%. ESI-MS: temperature. After 2 h, water (100 mL) was added and the solution
+
2+
m/z 1744.4 (calc 1743.9) [M+H] ; 872.2 (calc 872.9) [M+2H] ; MW was acidified to pH 2 with concentrated hydrochloric acid and then
1
742.85.
Synthesis of N-terminus E643-57-azide.
N-terminus E643-57-azide peptide
extracted with ethyl acetate (3 x 100 mL). The combined organic
layers were washed with water (2 x 50 mL) and brine (2 x 50 mL),
epitope
3 2 4
(N CH CO- dried over anhydrous MgSO and evaporated in vacuo to give sticky
QLLRREVYDFAFRDL) was synthesised following the general colourless solid. The crude product was purified with a preparative
procedure by manual stepwise SPPS HATU/DIPEA Fmoc-chemistry. RP-HPLC on C-18 column with a 25-45% solvent B gradient over 20
+
The attachment of azidoacetic acid (4.2 equiv.) was achieved using min. Yield: 23%. ESI-MS: m/z 418.5 (calc 418.5) [M+H] ; 835.6 (calc
+
+
HATU (3 equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h) 836.0) [2M+H] ; 1252.8 (calc 1253.4) [3M+H] ; MW 417.
and the reaction mixture was covered and protected from light with Synthesis of Pam2Cys-alkyne
aluminum foil. The crude product was purified by a preparative RP- Pam2Cys-alkyne was synthesised following the general manual
HPLC on C-18 column with a 35-55% solvent B gradient over 20 min. stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure (Scheme 6).
R
HPLC analysis (C-18 column, Method A): t = 23.3 min, purity > 95%. The attachment of Fmoc-Dhc-OH was achieved by dissolving a
2
+
Yield: 50%. ESI-MS: m/z 1012.8 (calc 1012.6) [M+2H] ; 675.7 (calc mixture of Fmoc-Dhc-OH (3 equiv.), DIC (3 equiv.) and HOBt (3
3
+
O
6
75.4) [M+3H] ; MW 2023.
equiv.) in DMF (2 mL) at 0 C for 5 min. The activated species was
Synthesis of lipid 1 conjugated with 8Qmin (27)
then added to the resin and left to couple for 4 h, followed by a
-3
A mixture of 8Qmin-azide (4.2 mg, 2 x 10 mmol, 1 equiv.) and the thorough wash with DMF and DCM. The S-glycerol-cysteine
-3
lipoalkyne 1 (1.7 mg, 3 x 10 mmol, 1.5 equiv.) was dissolved in hydroxyl groups were then palmitoylated by addition of palmitic
DMF (1 mL), and copper wire (80 mg) was added. The air in the acid (20 eq) activated with DIC (25 eq) and 4-
reaction mixture was removed by nitrogen bubbling. The reaction (dimethylamino)pyridine (DMAP; 2 eq) in DCM. The reaction was
1
0 | J. Name., 2012, 00, 1-3
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left to complete overnight, and the resin subsequently washed with powder. Analytical HPLC analysis (C-18 column, Method A) t = 18.3
R
DCM. The ε-Mtt protecting group on the C-terminal lysine was then min, purity > 95%. Yield: 1.2 mg, 92%. ESI-MS: m/z 1793.3 (calc
+
2+
removed by using a mixture of 1%TFA and 5% TIPS in DCM (25 x 1793.0) [M+H] ; 897.0 (calc 897.0) [M+2H] ; MW 1792.
min) followed by washing with DCM and DMF. The Dhc-associated
Fmoc group was then removed by using 2.5% (w/v) DBU in DMF (3 x Copper-Catalysed Alkyne-Azide Cycloaddition (CuAAC) Reaction -
min), followed by washing with DMF, DCM, and drying under model study for the synthesis of compound 16.
5
5
vacuum. The crude product was purified by a preparative RP-HPLC A mixture of model mercapto-acryloyl conjugation product 14 (1
on C-4 column with a 40-80% solvent B gradient over 60 min. HPLC equiv.) and 1,3-di(hydroxymethyl)-5-(prop-2-ynyloxy)benzene (15)
analysis (C-4 column, Method A): t = 33.5 min, purity > 95%. Yield: (1.5 equiv.) was dissolved in DMF (1 mL), and copper wire was
R
+
2
0%. ESI-MS: m/z 1351.2 (calc 1351.9) [M+H] ; 676.4 (calc 676.5) added. The air in the reaction mixture was removed by nitrogen
2
+
[M+2H] ; MW 1350.9.
bubbling. The reaction mixture was covered and protected from
light with aluminum foil and stirred at 50 °C under nitrogen. The
Synthesis of 8Qmin/E643-57-Pam2Cys (29)
A mixture of azide derivative (23) (3.9 mg, 8.8 x 10 mmol, 1 equiv.) progress of the reaction was monitored by analytical HPLC (C-18
and the Pam2Cys-alkyne (1.9 mg, 1.1 x 10 mmol, 1.2 equiv.) was column) until the model mercapto-acryloyl conjugate 14 was
-4
-3
dissolved in DMF (1 mL), and copper wire (80 mg) was added. The completely consumed after 1 h. Analytical HPLC analysis (C-18
air in the reaction mixture was removed by nitrogen bubbling. The column, Method A) for the model CuAAC product 16 t
R
= 17.5 min.
+
reaction mixture was covered and protected from light with ESI-MS: m/z 1985.5 (calc 1985.2) [M+H] ; 993.0 (calc 993.1)
2
+
aluminum foil and stirred at 50 °C under nitrogen. The progress of [M+2H] ; MW 1984.2.
the reaction was monitored by analytical HPLC (C-4 column) and Synthesis of N-terminal Model mercapto-azide (17).
ESI-MS until the peptide 23 was completely consumed after 5 h. N-terminal model mercapto-azide (N
3 2
CH CO-Cys-Lys-Gln-Ala-Glu-
The reaction mixture was purified using a semi-preparative HPLC on Asp-Phe-NH ) was synthesised following the general manual
2
a C-4 column (40-80% solvent B over 60 min). After lyophilization, stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. The
the pure 8Qmin/E643-57-Pam2Cys (29) was obtained as an amorphous attachment of azidoacetic acid (4.2 equiv.) was achieved using
white powder. Compound 29 was analysed by HPLC (C-4 column, HATU (3 equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h)
Method A) t
R
= 30.5 min, purity > 97% (detected by UV at 214 nm). and the reaction mixture was covered and protected from light with
4
+
Yield: (3.3 mg, 72%). ESI-MS: m/z 1298.8 (calc 1298.8) [M+4H] ; aluminum foil. The crude product was purified by a preparative RP-
5
+
6+
1
5
039.3 (calc 1039.2) [M+5H] ; 866.5 (calc 866.2) [M+6H] ; MW HPLC on C18 column with a 15-35% solvent B gradient over 20 min.
191.2. HPLC analysis (C18 column, Method A): t = 17.6 min, purity > 95%.
R
+
Yield: 69%. ESI-MS: m/z 922.6 (calc 922.4) [M+H] ; MW 922.0
Model Compounds
Synthesis of N-terminal acryloyl Model (18).
Synthesis of N-terminal Model mercapto-azide (12).
N-terminal acryloyl model
) was synthesised following the general manual
) was synthesised following the general manual stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. The
2
(CH =CHCO-Phe-Ala-Ala-Lys-Lys-
N-terminal model mercapto-azide (N
Asp-Phe-NH
3 2 2
CH CO-Cys-Gln-Ala-Glu-Pro- Cys(Acm)-NH
2
stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. The coupling of acrylic acid (4.2 equiv.) was achieved using HATU (4
attachment of azidoacetic acid (4.2 equiv.) was achieved using equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h). The crude
HATU (3 equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h) product was purified by a preparative RP-HPLC on C18 column with
and the reaction mixture was covered and protected from light with a 20-40% solvent B gradient over 20 min. HPLC analysis (C18
aluminum foil. The crude product was purified by a preparative RP- column, Method A): t
R R
= 14.1 min and (C4 column, Method A): t =
HPLC on C18 column with a 20-40% solvent B gradient over 20 min. 8.4 min, purity > 95%. Yield: 27%. ESI-MS: m/z 791.5 (calc 791.4)
+
R
HPLC analysis (C18 column, Method A): t = 15.9 min, purity > 95%. [M+H] ; MW 791.
+
Yield: 60%. ESI-MS: m/z 891.8 (calc 891.9) [M+H] ; MW 890.9.
One pot double conjugation reaction to synthesise 20
Synthesis of N-terminal acryloyl Model (13).
A mixture of N-terminal model mercapto-azide 17 (2.0 mg, 2.0
The N-terminal acryloyl model peptide (CH
2
=CHCO-Gln-Leu-Leu- µmol, 2 equiv.) and N-terminal acryloyl model 18 (1.0 mg, 1.0 µmol,
Arg-Arg-Tyr-NH ) was synthesised following the general manual 1.0 equiv.) was dissolved in a guanidine buffer (1 mL) at ~pH 7.3.
2
stepwise SPPS HATU/DIPEA Fmoc-chemistry procedure. The The reaction mixture was incubated at 37 °C for 72 h. The progress
coupling of acrylic acid (4.2 equiv.) was achieved using HATU (4 of the reaction was monitored by analytical HPLC until the N-
equiv.)/DIPEA (4.2 equiv.) at room temperature (2 × 1 h). The crude terminus model mercapto azide 17 was completely consumed and
product was purified by a preparative RP-HPLC on C18 column with the model conjugate azide (19) was formed. A mixture of 1,3-
a 20-40% solvent B gradient over 20 min. HPLC analysis (C18 di(hydroxymethyl)-5-(prop-2-ynyloxy)benzene (15) (1.92 mg, 10
column, Method A): t
R
= 17.7 min, purity > 95%. Yield: 40%. ESI-MS: µmol, 10 equiv.) in DMF:DMSO (1:1) (0.5 mL) and copper wire (60
+
2+
m/z 901.9 (calc 902.1) [M+H] ; 451.4 (calc 451.5) [M+2H] ; MW mg) was added to the reaction mixture. The air in the reaction
9
01.1.
mixture was removed by nitrogen bubbling. The reaction mixture
was stirred at 50 °C under nitrogen. The progress of the reaction
Synthesis of Model mercapto-acryloyl conjugation product (14)
A mixture of N-terminal model mercapto-azide 12 (1.1 mg, 1.2 was monitored by analytical HPLC (C-18 column) until 19 was
µmol, 2 equiv.) and N-terminal acryloyl model peptide 13 (0.7 mg, completely consumed after 1 h and gave the final product 20.
+
0
.6 µmol, 1.0 equiv.) was dissolved in a guanidine buffer at ~pH 7.3. ESI-MS of compound 19: m/z 1713.2 (calc 1714.0) [M+H] ; 857.1
2
+
3+
The reaction mixture was incubated at 37 °C for 14 h. The progress (calc 857.5) [M+2H] ; 571.7 (calc 572.0) [M+3H] ; MW 1713.
2
+
of the reaction was monitored by analytical HPLC until the N- ESI-MS of compound 20: m/z 953.7 (calc 953.6) [M+2H] ; 636.2
3
+
terminus acryloyl model was completely consumed. The reaction (calc 636.1) [M+3H] ; MW 1905.
mixture was purified using a semi-preparative HPLC on a C-18 Particle size measurement
column (10-50% solvent B over 60 min). After lyophilization, the The final constructs of lipopeptides 24-26 and 29 were dissolved in
pure azide product 14 was obtained as an amorphous white sterile PBS using a vortex until a homogenous solution was
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obtained. The particle sizes were measured by dynamic light µg/ml; clone R4-6A2; eBioscience) in 1% BSA added at room
scattering. The measurements were repeated at least five times. All temperature for 3 hours. After washing, a streptavidin-HRP complex
compounds formed particles with rather high polydispersity. (DakoCytomation) in 1% BSA was added for 1 h at room
Compound 24 and 25 formed submicron particles (24: 450 – 750 temperature, plates were washed again, and bound cytokine was
nm, PDI = 0.25 – 0.55 and 25: 350 – 550 nm, PDI is 0.20 – 0.40). visualised with 3-amino-9-ethylcarbozole (Calbiochem). Spots were
Compounds 26 and 29 formed large aggregates (visible to the counted with an ELISPOT reader (Autoimmun Diagnostika). An
naked eye) with sizes above the upper detection level of the “irrelevant” lipopeptide (KQAEDKVKASREAKKQVEKALEQLEDKVK -
instrument (>5 µm).
conjugated with lipid 1) was used as a control in this experiment.
Biological Assay
Group A streptococcal B-cell epitope (J14) was selected as the
3
1
Mice and Cell Lines. Female C57BL/6 (6−8 weeks old) mice were irrelevant peptide.
used in this study and purchased from Animal Resources Centre
(Perth, Western Australia). TC-1 cells (murine C57BL/6 lung Statistical Analysis. All data were analysed using GraphPad Prism 5
epithelial cells transformed with HPV-16 E6/E7 and ras software. Kaplan−Meier survival curves for tumour treatment
2
9
O
oncogenes). TC-1 cells were cultured and maintained at 37 C/5% experiments were applied. Differences in survival treatments were
CO in RPMI 1640 medium (Gibco) supplemented with 10% heat determined using the log-rank (Mantel-Cox) test, with p < 0.05
2
inactivated fetal bovine serum (Gibco) and 1% nonessential amino considered statistically significant.
acid (Sigma-Aldrich). The animal experiments were approved by the
University
of
Queensland
Animal
Ethics
committee
Acknowledgements
(DI/034/11/NHMRC) and (UQDI/327/13/NHMRC) in accordance
with National Health and Medical research Council (NHMRC) of
Australia guidelines.
Tumor challenge experiments. To test the efficacy of lipopeptide
This work was supported by the National Health and Medical
Research Council of Australia (NHMRC 1006454). We thank Dr.
Zeinab Khalil from the Institute for Molecular Bioscience, The
University of Queensland for her assistance in acquiring the
high-resolution mass spectroscopy data. We thank Thalia
Guerin for her critical review of this manuscript.
24 conjugate as a therapeutic vaccine against established tumours,
groups of C57BL/6 female mice (8/group) were first challenged
5
subcutaneously in the right flank with 1 × 10 /mouse of TC-1
tumour cells. On the third day after tumour challenge, the mice
were injected subcutaneously at the tail base with: 100 μg of
lipopeptide 24-26 conjugates in a total volume of 100 μL of sterile-
filtered PBS; a mixture of lipid 1 conjugated with 8Qmin (27) and lipid
References
1
conjugated with E643-57 (28) (100 μg/100 μL sterile PBS, 1:1);
1
(a) A. W. Purcell, J. McCluskey and J. Rossjohn, Nat. Rev.
Drug Discovery, 2007, , 404-414; (b) J. Pol, N. Bloy, A.
8Qmin/E643-57-Pam2Cys (29) (100 μg/100 μL sterile PBS) as a positive
6
control; or 100 μL PBS as a negative control. Each mouse received a
single immunization only. The size of the tumour was measured by
palpation and calipers every two days and reported as the average
tumour size across the group of five mice or as tumour size in
Buque, A. Eggermont, I. Cremer, C. Sautes-Fridman, J. Galon,
E. Tartour, L. Zitvogel, G. Kroemer and L. Galluzzi,
Oncoimmunology, 2015, 4.
2
3
4
M. Skwarczynski and I. Toth, Nanomedicine, 2014, 9, 2657-
2669.
S. G. Reed, S. Bertholet, R. N. Coler and M. Friede, Trends
Immunol., 2009, 30, 23-32.
R. Rappuoli, M. Pizza, G. Del Giudice and E. De Gregorio,
Proceedings of the National Academy of Sciences of the
United States of America, 2014, 111, 12288-12293.
(a) F. Azmi, A. A. A. Fuaad, M. Skwarczynski and I. Toth,
Human Vaccines & Immunotherapeutics, 2014, 10, 778-796;
3
0
individual mice. Tumour volume was calculated using the formula
3
2
30b
V (cm ) = 3.14 × [largest diameter × (perpendicular diameter) ]/6.
3
The mice were euthanised when tumour reached 1 cm or started
bleeding to avoid unnecessary suffering.
The second tumor challenge study was executed with 24 and
antigens administered with commercial adjuvant, in the similar
manner as described above. Two independent experiments were
performed with total 13 C57BL/6 mice per group (5 + 8 mice per
group) and the list of the immunization compounds was:
lipopeptide 24; 30 μg of a mixture of 8Qmin and E643-57 emulsified in
a total volume of 100 μL of Montanide ISA51 (Seppic, France)/PBS
5
6
(
b) R. J. Hopkins, N. F. Daczkowski, P. E. Kaptur, D. Muse, E.
Sheldon, C. LaForce, S. Sari, T. L. Rudge and E. Bernton,
Vaccine, 2013, 31, 3051-3058.
(a) P. M. Moyle, C. Olive, M.-F. Ho, M. F. Good and I. Toth, J.
Med. Chem., 2006, 49, 6364-6370; (b) M. Skwarczynski, M.
Zaman, C. N. Urbani, I. C. Lin, Z. Jia, M. R. Batzloff, M. F.
Good, M. J. Monteiro and I. Toth, Angew. Chem., Int. Ed.,
(1:1, v/v) as a positive control; and PBS as a negative control.
IFN-gamma ELISPOT assays
Splenocytes were harvested from the spleens of naïve and
immunised mice and depleted of red-blood cells using Red Blood
Cell Lysing Buffer (0.155 M ammonium chloride in 0.01 M Tris-HCl
buffer, Sigma). Splenocytes were then resuspended in RPMI (Sigma)
supplemented with 20% FCS (Sigma), 100 U/ml penicillin and 100
µg/ml streptomycin, and 50 µM β-mercaptoethanol. The cells were
2
010, 49, 5742-5745; (c) M. Black, A. Trent, Y. Kostenko, J. S.
Lee, C. Olive and M. Tirrell, Adv. Mater. , 2012, 24, 3845-
849; (d) T. H. Wright, A. E. S. Brooks, A. J. Didsbury, G. M.
3
Williams, P. W. R. Harris, P. R. Dunbar and M. A. Brimble,
Angew. Chem., Int. Ed., 2013, 52, 10616-10619; (e) F. Boato,
R. M. Thomas, A. Ghasparian, A. Freund-Renard, K. Moehle
and J. A. Robinson, Angew. Chem., Int. Ed., 2007, 46, 9015-
9018.
5
plated at 5x10 cells/well in triplicate in ELISPOT plates (Millipore
Biotec) which had been previously coated with 5 µg/ml IFN-γ
O
C
capture mAb (clone 14-7313-85 eBioscience) in PBS at 4
7
8
H.-J. Cho, Y.-K. Oh and Y. B. Kim, Expert Opin. Ther. Pat.,
2011, 21, 295-309.
O. Peralta-Zaragoza, V. H. Bermudez-Morales, C. Perez-
Plasencia, J. Salazar-Leon, C. Gomez-Ceron and V. Madrid-
Marina, Onco Targets Ther, 2012, 5, 315-328.
overnight and then blocked with RPMI/20% FCS at room
temperature for 3 hours. E7 (RAHYNIVTF) and E6 (YDFAFRDL)
peptides (10 µg/ml) were added alongside 10 ng/ml rhIL-2 (R&D
systems) to a final volume of 200 µl/well. Plates were incubated for
O
1
8 hours at 37 C, washed, and a biotinylated IFN-γ detection Ab (2
1
2 | J. Name., 2012, 00, 1-3
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