Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope

Article abstract of DOI:10.1016/j.bmc.2014.06.052

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund's Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.

Full text of DOI:10.1016/j.bmc.2014.06.052

Bioorganic & Medicinal Chemistry 22 (2014) 4848–4854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Active immunisation of mice with GnRH lipopeptide vaccine
candidates: Importance of T helper or multi-dimer GnRH epitope
Daryn Goodwin a, Pavla Simerska a, Cheng-Hung Chang a, Friederike M. Mansfeld a, Pegah Varamini a,
Michael J. D’Occhio b, Istvan Toth a,c,
⇑
a The School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Queensland, Australia
b The Faculty of Agriculture and Environment, The University Sydney, Camden 2570, New South Wales, Australia
c The School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba 4102, Queensland, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgi-
cal castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of
vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH pep-
tide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope,
was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to
determine the minimal components of this vaccine necessary to induce a systemic immune response.
BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Com-
plete Freund’s Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres
were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an
increase in titres observed after successive immunisations. This study highlights the importance of for
epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo.
The results of this study also contribute to the development of future clinical and veterinary
immunocontraceptives.
Received 11 April 2014
Revised 13 June 2014
Accepted 23 June 2014
Available online 1 July 2014
Keywords:
Gonadotropin releasing hormone
Lipopeptide
Vaccine
T helper
Murine immunogenicity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
In the 1970s a number of groups attempted to produce antibod-
ies against GnRH by co-administration with Complete Freund’s
Our understanding and approaches to block or limit the release
of reproductive hormones in an attempt to limit their influence on
cancers has progressed to the development of immunotherapies. In
spite of low immunogenicity, active immunisation against gonado-
tropin releasing hormone (GnRH) has received considerable atten-
tion because of potential applications in immunocontraception. An
active area of research is the development of synthetic GnRH-
based vaccines against reproductive hormone-dependent male
and female cancers.1 Studies have shown that immunisation
against GnRH can reduce prostate levels of testosterone similar
to surgical castration in numerous mammalian models, including
mice and humans.2,3 Immunisation of both males and females
against GnRH can have a profound effect on fertility through the
reduction in sex steroids and cessation of gametogenesis. Hence,
there is a potential for GnRH therapeutics to be used either as a
semi-permanent contraceptive or to extend the postnatal anovula-
tory period.4,5
Adjuvant (CFA). However, these attempts were unsuccessful in
producing high anti-GnRH antibody titers.6 Many small molecules
like peptides (haptens) are successful immunogens only if they are
attached to macromolecules (carriers). It is often necessary to
modify these haptens for coupling with carriers to make a stable
carrier–hapten complex.7 Selection of a suitable carrier system,
hapten density (hapten:carrier molar ratio), and conjugation meth-
ods are critical for developing an optimal vaccine against hap-
tens.8,9 The first alum-adjuvanted vaccine which was successful
in producing antibodies against GnRH was conjugated to a tetanus
toxoid carrier.10 A number of other fusion proteins have been pre-
pared for the production of anti-GnRH antibodies that employ con-
jugation to diphtheria toxoid (e.g. ImprovacÒ), keyhole limpet
hemocyanin (e.g. GonaCon™) or ovalbumin, with many of them
available commercially for veterinary use.11–15 Another recombi-
nant fusion protein consisting of GnRH and diphtheria toxoid
was successful in phase I/II clinical trials.16
Synthetic subunit vaccine systems eliminate the need for large
carrier proteins, which are often associated with adverse effects in
the host.17 Since subunit vaccines are composed of different
⇑
Corresponding author. Tel.: +61 7 33469892; fax: +61 7 33654273.
E-mail address: i.toth@uq.edu.au (I. Toth).
http://dx.doi.org/10.1016/j.bmc.2014.06.052
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. Goodwin et al. / Bioorg. Med. Chem. 22 (2014) 4848–4854
4849
individual components (i.e. epitopes, carriers, adjuvants), they are
highly customisable. This makes them valuable tools for develop-
ing vaccines against weak immunogens, such as GnRH.18 Lipopep-
tides can form a synthetic subunit vaccine as one entity that
includes all components (adjuvant, carrier and antigen) required
by a successful vaccine, eliminating the need for co-administration
with an adjuvant.19,20
High antibody titers were generated when multiple copies of a
peptide were incorporated into a multiple antigenic peptide sys-
tem with a branched polylysine core.20 Immunogenicity was fur-
ther enhanced by the incorporation of lipoamino acid (LAA)
residues with this polylysine system, which led to the develop-
ment of novel candidates for vaccines against a variety of disease
states, including cancer. The lipid moieties in the lipopeptide
system serve to mimic the action of bacterial adjuvants such as
Pam3Cys and Pam2Cys.21 Lipopeptide constructs have been found
to mediate significant immunogenic effects without additional
adjuvants.22,23
protected aminopropyl groups as attachment points for GnRH pep-
tide epitopes.27,28
The first series of compounds (1–3, Fig. 1) contained four copies
of the GnRH peptide epitope with three copies of 12-carbon LAAs
(C12) separated by glycine spacers (1 and 2) or 16-carbon LAAs
(C16) (3) attached to either a poly-lysine (1 and 3) or onto a glu-
cose core (2). GnRH-based vaccine candidates bearing Pam2Cys
adjuvanting lipid moiety and T helper epitope derived from the L
chain of influenza virus hemagglutinin (GALNNRFQIKGVELKS)
was effective in a female mouse model.29 A similar construct (4)
consisting of two C16 lipid moieties was therefore used in this
study. To investigate the immunogenic potential of the GnRH epi-
tope, four copies of a tandem GnRH dimer were synthesised on a
poly-lysine lipopeptide to yield 8 copies of the epitope in total
(5). The presentation of GnRH was enhanced by replacing the Gly
residue at position 6 in the parent peptide sequence with a D-Cys
(pEHWSYcLRPG30 and conjugated to a linear T helper lipopeptide
(60) using Michael addition (Fig. 2).31 The site-specific conjugation
allowed for both terminal regions of GnRH to display the free
amino groups important for immune system recognition (6). The
lipopeptide was designed to have only two 16-carbon LAAs at
the N-terminus, with an additional Ser spacer for improved
solubility. These modifications led to improved yields (70%) com-
pared to the original design that incorporated a lipidic tail similar
to 3 (4%). In compounds 4–6, serine residues were used as
spacers between the lipid moiety and epitopes, a modification that
was previously reported to enhance the immune response of
lipopeptides.32
The development of a GnRH-based subunit lipopeptide vaccine
for use as an immunocontraceptive therapy has previously
received attention.24 In this study, we aimed to define the minimal
components necessary for the induction of a systemic immune
response against GnRH. The immunogenicity of the GnRH deca-
peptide (EHWSYGLRPG) was enhanced by incorporating it into
the lipopeptide systems.
2. Results and discussion
2.1. Peptide synthesis
2.2. In vitro peripheral blood mononuclear cell (PBMC) toxicity
assay
Lipopeptides 1–5 (Fig. 1) were synthesised by manual and
microwave-assisted solid-phase protocols with Boc chemistry.25
Lipidic moieties were prepared from their 1-bromoalkanes using
the method of Gibbons et al. and were used as a mixture of enan-
tiomers in subsequent synthesis.26 Amino acid couplings involved
an activation of residues with DIPEA in DMF, followed by in situ
neutralization in the presence of HATU or HBTU. The carbohydrate
Numerous methods exist to evaluate cellular toxicity; most of
them use cell cultures to screen for toxicity by either observing
changes in normal cell function or detecting dead cells with
stain-based assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium (MTT) bromide assay has been widely used for deter-
mining the cytotoxicity of compounds.33 We adopted this
method with the peripheral blood mononuclear cells (PBMCs) as
a model to evaluate the cytotoxicity of the library of lipopeptide
vaccine candidates. This model is particularly relevant for testing
of drug candidates, and for the assessment of potential toxic risks
associated with clinical trials.
core for vaccine construct 2 was synthesised from a
D-glucose
derivative that bore an adipate linker and four tert-butoxycarbonyl
R2
R1
GnRH*
In this assay, vaccine candidates 1–6 were tested at four rising
concentrations (10, 50, 100 and 200 lM). A slight decrease in cell
viability was observed by the highest concentration of the vaccine
constructs after 48 h; however, it was not statistically significant
compared to the negative control (Fig. 3, p >0.05).
1:
2:
3:
(K)3 C12-G-C12-C12-G
Glc C12-G-C12-C12-G
(K)3 C16-G-C16-C16-G
GnRH*
GnRH*
R1-R2-NH2
GnRH*
R2
-NH2
T helper epitope
GnRH
Lys
R2
4:
5:
S-S-C16-C16
GnRH*
GnRH
O
R2
(K)3 S-S-C16-C16
R1
[
D
-Cys6]GnRH*
SH
-R2-NH2
N
O
T helper epitope
GnRH*
GnRH*
GnRH
GnRH
+
R1-R2-NH2
6'
10% DMF/PBS
pH 7.4
GnRH*
GnRH
R2
[D
-Cys6]GnRH*
[
D
-Cys6]GnRH*
6:
S-C16-C16
O
S
-R2-NH2
T helper epitope
-R2-NH2
N
T helper epitope
O
R2 = S-C16-C16
6
GnRH = EHWSYGLRPG
GnRH* = pEHWSYGLRPG
T helper epitope = GALNNRFQIKGVELKS
Figure 2. Conjugation of [
form 6.
D
-Cys6]GnRH⁄ to maleimide conjugated adjuvant (60), to
Figure 1. Structure of GnRH lipopeptide subunit vaccines (1–6).

4850
D. Goodwin et al. / Bioorg. Med. Chem. 22 (2014) 4848–4854
Figure 3. Percentage of PBMC viability compared to phosphate-buffered saline (PBS) after 48 h incubation with the lipopeptide vaccine candidates 1–6 at four concentrations.
Data represented as mean SEM. Statistical analysis was performed using one-way ANOVA followed by the Dunnett’s post hoc test and compared to the PBS group.
2.3. Murine immunogenicity of synthetic GnRH vaccine
candidates
compound 4 administered with CFA was the only compound to
produce significant GnRH-specific IgG antibody titers (Fig. 4). This
demonstrated that the lipopeptide system alone was insufficient to
adjuvant GnRH and required the incorporation of the T helper epi-
The first study was performed to determine the ability of the
GnRH lipopeptides to induce an immune response when four cop-
ies of GnRH were incorporated into polylysine or carbohydrate
scaffolds (1–3). Compounds 1–3 without T helper epitope were
compared to compound 4, which consisted of one copy of GnRH
along with a T helper epitope. All lipopeptide vaccine candidates
tope or another strategy to stimulate
a significant immune
response in this model. To this end we designed two additional
compounds to improve the immunogenicity of the anti-GnRH vac-
cine construct.
Lipidated GnRH dimers have previously been shown to elicit an
antibody response in murine models when administered with
CFA.31 Therefore, in order to increase the possibility of inducing
an immune response to GnRH, the peptide was dimerised in tan-
dem and attached to the lipopeptide system in four copies (8 GnRH
epitopes in total; 5). The vaccine 5 co-administered with CFA pro-
duced IgG titers comparable to the positive control 4 over the
course of the study. Notably, when mice were administered with
5 alone (no CFA), comparable antibody titres (p >0.05) to 5 with
CFA were achieved at day 63, (Fig. 5). This demonstrated the adju-
vanting activity of the lipopeptide in the construct. It is plausible
that dimerization of the GnRH epitope in the construct induced
an immune response either due to the formation of small receptor
were dissolved in PBS at a concentration of 1 lg l
Lꢀ1 and adminis-
tered with CFA to adjuvant a significant response to each construct.
It was proposed that the immunogenicity of the GnRH hapten
would benefit from increased molecular weight, increased copy
number (possible B-cell receptor clustering), and the incorporation
of a lipid adjuvant. It was anticipated that GnRH linked by the
lysine residues of the polylysine core (1 and 3) would produce a
structure that was large enough to be recognised and processed
by the immune system. Since the spatial arrangement may play a
part in the immunogenicity of synthetic subunit vaccines, a carbo-
hydrate scaffold was used as a peptide antigen carrier to assemble
2. However, the first study using compounds 1–4 showed that
6
ns
6
4
2
0
***
***
***
**
***
4
2
0
Figure 5. GnRH-specific serum IgG titers (log10) at day 63 after primary immuni-
sation in response to intramuscular immunisation of BALB/c mice as determined by
ELISA for individual sera from the second study (4–6). Mean IgG titers are presented
as a bar. Primary immunisation on day 0 was followed by boosts on days 28 and 56.
Statistical analysis was performed using a one-way ANOVA followed by the Tukey
Figure 4. GnRH-specific serum IgG titers (log10) at day 64 after primary immuni-
sation in response to intramuscular immunisation of BALB/c mice as determined by
ELISA of individual sera from the first study (1–4). Mean IgG titers are represented
by a bar. Primary immunisation on day 0 was followed by boosts on days 28 and 56.
Statistical analysis was performed using a one-way ANOVA followed by the Tukey
post hoc test and compared to the PBS group (⁄⁄⁄p <0.001).
post hoc test, when each compound was compared to the PBS group (⁄⁄p <0.01; ⁄⁄⁄
<0.001) or when 5 with CFA (5 + CFA) was compared to 5 alone (ns, p >0.05).
p

D. Goodwin et al. / Bioorg. Med. Chem. 22 (2014) 4848–4854
4851
clusters co-localised by binding of polyvalent antigens or by the
creation of epitopes in the junction of the dimer.34 In a previous
study multiple copies of GnRH was added to a fragment of human
IgG and T helper epitope derived from measles virus. It was shown
that a significantly greater antibody response was elicited com-
pared to that with same sequence but only a single copy of GnRH
epitope.35
Interestingly, the vaccine candidate 6 in PBS induced slightly
higher GnRH-specific IgG titers in mice than 4 with CFA (Fig. 5).
Both constructs consisted of identical principal components
(GnRH, T helper epitope and lipid adjuvant), yet arranged in differ-
ent configurations and conjugated through alternate bonds and
positions. Our findings are consistent with a previous study where
thioester linkages elicited higher antibody titers when compared
to a library of alternative bonds.36 It was suggested that different
spatial arrangements of antigen, carrier, and adjuvant, and the
presence of different bonds were responsible for variation in
antibody titers.
medium (DMEM), Hank’s balanced salt solution (HBSS), 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) and PBS
were obtained from GIBCO (Mulgrave, VIC, Australia). Isopropanol
was sourced from Lab-Scan Pty Ltd (Dublin, Ireland). All other
reagents were purchased from Sigma–Aldrich (Castle Hill, NSW,
Australia).
A Kel-F HF apparatus (Peptide Institute, Osaka, Japan) was used
for HF cleavage. Thin layer chromatography (TLC) was carried out
on Kieselgel 60 F254 silica gel coated aluminum plates (Merck,
Darmstadt, Germany). All TLCs were visualised by 20% H2SO4 or
p-anisaldehyde followed by heating. Tetrahydrofuran (THF), DCM
and CHCl3 were dried by distillation with sodium/benzophenone,
calcium hydride and calcium chloride, respectively, then used
immediately or stored with 4 Å molecular sieves.
1H NMR spectra were recorded on a BRUKER Avance spectrom-
eter at 300 MHz or 500 MHz in CDCl3. ESI-MS was performed on a
triple quadrupole Perkin–Elmer-Sciex API3000 mass spectrometer
using a solvent mixture of A [H2O + 0.1% AcOH] and B [MeCN/
H2O + 0.1% AcOH]. Data were acquired and analysed using Analyst
1.4 (Applied Biosystems/MDS Sciex, Toronto, Canada) software.
Reverse-phase-high performance liquid chromatography (RP-
HPLC) analysis was carried out using a Shimadzu instrument
(Kyoto, Japan), with a Grace Vydac (Columbia, MD, USA) Protein/
Peptide column. Analysis was achieved using a gradient of solvent
A (99.9% H2O/0.1% TFA) and solvent B (90% MeCN/H2O/0.1% TFA). A
gradient of 0–100% solvent B (C4), 40–70% solvent B (C40), or 40–
80% solvent B (C400) over 30 min was employed and absorbance
detected at 214 nm. Crude peptides were then purified using a
Waters Delta Prep 600 (Milford, MA, USA) or Shimadzu (LC-20AT,
SIL-10A, CBM-20A, SPD-20AV, FRC-10A; 20 mL minꢀ1) preparative
RP-HPLC with a Grace Vydac C4/C18 column. Separation was
achieved using a gradient of solvent A and solvent B at a flow rate
of 10 mL minꢀ1 detected at 230 nm. All peptides were character-
ised using ESI-qQTOF on an Applied Biosystems QSTAR Pulsar mass
spectrometer (Foster City, CA, USA) or ESI-MS on a triple quadru-
pole API 3000 (Sciex/Applied Biosystems).
3. Conclusion
This study outlines the synthesis, cytotoxicity and immunoge-
nicity of a library of novel lipopeptide subunit vaccines for GnRH.
Self-adjuvanting lipopeptide GnRH vaccine candidates that con-
tained multiple GnRH monomer/dimer epitopes or a thioester-
linked GnRH helper epitope system were identified and developed.
The investigation of the structure–activity relationship and opti-
mal epitope configuration reinforced that the inclusion of both B
and
T helper epitopes has a significant effect on systemic
anti-GnRH IgG titers. It was also demonstrated that alternative
strategies, such as using multiple copies of a peptide dimer, were
successful without the need for additional helper epitopes, which
can be useful because helper epitope selection can be challenging,
particularly when developing a vaccine to be tested on cross-
species platforms.37,38
This study confirmed the self-adjuvanting activity of the LAA
moieties by eliciting comparable anti-GnRH IgG titers to the titers
obtained from the constructs co-administered with CFA. Further
analysis is necessary to optimise and understand the mechanisms
responsible for the immunogenicity of these lipopeptide vaccine
candidates and their epitopes. These findings significantly advance
out understanding of anti-hapten subunit vaccine design and could
contribute to the future development of a commercially viable sub-
unit-based GnRH vaccine.
4.2. Chemistry
4.2.1. 6-Maleimidocaproic acid39
Maleic anhydride (2.94 g, 30 mmol) and 6-aminocaproic acid
(3.94 g, 30 mmol) were refluxed in AcOH (70 mL) overnight. Ac2O
(2.83 mL, 30 mmol) was added dropwise and reflux continued for
a further 1 h. The acetic acid was co-evaporated with toluene in
vacuo to yield yellow syrup. The material was purified over SiO2
(DCM:MeOH, 10:0.7) affording a cream crystalline solid in 57%
yield (3.58 g): Rf (DCM/MeOH, 10:0.7) = 0.50; ESI-MS (C10H13NO4,
211.1): m/z = 212.0 [M+H+] (calcd 212.1), 423.1 [M+2H+] (calcd
423.2); 1H NMR: d = 6.95 (2H, s, CH@CH), 3.35 (2H, t, J 7.4 Hz,
NCH2), 2.15 (2H, t, J 7.2 Hz, CH2COOꢀ), 1.45 (4H, m, 2CH2), 1.18
(2H, m, CH2).
4. Experimental section
4.1. Materials and methods
Unless otherwise stated, all chemicals were of analytical grade
or equivalent. Acetic anhydride (Ac2O) was supplied by Univar
(Ingleburn, NSW, Australia). N,N-Dimethylformamide (DMF) was
obtained from EMD (Darmstadt, Germany). HATU, HBTU,
4.2.2. 2-tert-Butoxycarbonylaminododecanoic acid lipoamino
acid (Boc-C12-OH)40
Boc- and Fmoc-L-amino acids, and resins were obtained from
Mimotopes (Clayton, VIC, Australia), Novabiochem (Läufelfingen,
Switzerland) and Peptides International (Louisville, Kentucky).
Di-tert-butyldicarbonate was purchased from Auspep (Melbourne,
VIC, Australia). Acetic acid (AcOH), N,N-dichloromethane (DCM),
diethyl ether, diisopropylethylamine (DIPEA), HCl, hydrogen bro-
mide in AcOH, ninhydrin, triethylamine (TEA) and trifluoroacetic
acid (TFA) were supplied by Merck (Kilsyth, VIC, Australia). HPLC
and MS grade acetonitrile (MeCN) and methanol (MeOH) were
supplied by Scharlau (Port Adelaide, SA, Australia). Anhydrous
hydrofluoric acid (HF) was supplied by BOC gases (Sydney, NSW,
Australia). For in vivo experiments, Dulbecco’s modified Eagle’s
2-Aminododecanoic acid was synthesised as described in the
literature with 1-bromodecane and diethyl acetoamidomalonate.26
The free amine of 2-aminohexadecanoic acid was then Boc-pro-
tected by reaction with di-tert-butyl dicarbonate in a basic envi-
ronment as previously reported.26,41 C12 was prepared to afford
the pure product in 54.0% yield (1.31 g): Rf = 0.65 (DCM/MeCN/
AcOH); ESI-MS (C17H33NO4, 315.2): m/z = 316.4 [M+H]+ (calcd
316.2), 338.2 [M+Na]+ (calcd 338.2); 1H NMR: d = 4.94–4.92 (1H,
d, J 7.1 Hz, OCONH), 4.26 (1H, m, a-CH), 1.82–1.67 (2H, m, b-CH),
1.42 (9H, s, C(CH3)3), 1.23 (16H, br s, 8CH2), 0.86 (3H, t, J 7.0 Hz,
CH3).

4852
D. Goodwin et al. / Bioorg. Med. Chem. 22 (2014) 4848–4854
4.2.3. 2-tert-Butoxycarbonylaminohexadecanoic acid lipoamino
acid (Boc-C16-OH)42
formyl/Fmoc and DNP protecting groups were removed with 20%
piperidine in DMF and 20% 2-mercaptoethanol/10% DIPEA in
DMF, respectively. The resin was washed with DMF after each
manipulation. After the last coupling, all terminal Boc and Fmoc
groups were removed. The resin was then washed consecutively
with DMF, DCM and MeOH and left to dry in vacuo overnight.
The peptides were cleaved from the resin using the high HF
method at a concentration of 10 mL gꢀ1 of resin and 5% p-cresol
at ꢀ5 to 0 °C for 1–2 h. Following this, the peptide was precipitated
in diethyl ether, washed through a polyethylene frit, then dissolved
in 1:1 MeCN/H2O and lyophilised.
2-Aminohexadecanoic acid was synthesised as described in the
literature with 1-bromotetradecane and diethyl acetoamidomalo-
nate. The free amine of 2-aminohexadecanoic acid was then
Boc-protected by reaction with di-tert-butyl dicarbonate in a basic
environment as previously reported.26 The C16 product was pre-
pared to yield (78%) the pure product (1.89 g): Rf = 0.68 (DCM/
MeCN/AcOH); ESI-MS (C21H41NO4, 371.6): m/z = 394.3 [M+Na]+
(calcd 374.6); NMR: d = 4.92 (1H, m, OCONH), 4.26 (1H, m,
a-CH), 1.93–1.58 (2H, m, J 7.5 Hz, b-CH), 1.45 (9H, s, C(CH3)3),
1.25 (24H, br s, 12CH2), 0.86 (3H, t, J 6.9 Hz, CH3).
Synthetic adjuvants for conjugation to [D
-Cys6]GnRH were
assembled using microwave-assisted Fmoc solid phase peptide
synthetic protocols. The peptides were synthesised on Rink Amide
MBHA LL (100–200 mesh, 0.34 mmol gꢀ1) resin (Peptides Interna-
tional, USA), which was swelled in DMF/DIPEA for approximately
15 min. Peptides were then coupled using amino acids that had
been activated for 1 min with an equimolar amount of 0.5 M HATU
in DMF and 5 equiv DIPEA, then mixed with the resin for 10 min at
70 °C, 20 W. The coupling efficiencies were monitored using the
ninhydrin test calculated by reading the absorbance at 570 nm. If
the coupling efficiency was below 99.6%, the coupling was repeated
until the required efficiency was achieved. For Fmoc chemistry syn-
4.2.4. 2-((1-(4,4-Dimethyl-2,6-
dioxocyclohexylidene)ethyl)amino)hexadecanoic acid (Dde-
C16-OH)43
5,5-Dimethyl-1,3-cyclohexanedione (2.5 g, 17.8 mmol) was dis-
solved in DCM (15 mL). 4-Dimethylaminopyridine (435 mg) and
TEA (5 mL) were then added and the mixture was stirred for
10 min. Ac2O (2.2 mL) was added and the mixture was stirred
under Ar for 2 days. The solvent was removed in vacuo by co-evap-
oration of the crude product in ethyl acetate (EtOAc) (50 mL) with
toluene and then washed with 5% HCl (3 ꢁ 50 mL) and dried with
MgSO4. The solvent was evaporated to produce an oil which was
filtered through a column of silica (Hex/EtOAc, 3:2). The EtOAc
from the mixture obtained was evaporated in vacuo to afford an
orange oil, which was then cooled to yield pale yellow crystals
(2-acetyldimedone, Dde-OH) in 72% yield (2.35 g): Rf (Hex:EtOAc,
3:2) = 0.76; ESI-MS (C10H15NO2, 181.1): m/z = 182.2 [M+H]+ (calcd
182.1); 1H NMR: d = 2.59 (s, 3H, C@C(CH3)2) ; 2.52, 2.35 (2s, 4H,
2CH2) ; 1.06 (s, 6H, 2CH3).
thesis, Fmoc protecting groups were used for the a-amino-termini;
Pbf for Arg; Trt for Asn, Cys, His, and Gln; tBu for Ser; Boc for Trp and
Lys; and OtBu for Glu. Dde-C16-OH was used to assemble the N-ter-
minal lipid adjuvant. The Fmoc protecting groups were removed
from the amino acids using 20% piperidine/DMF. Dde groups were
removed using 2% hydrazine hydrate in DMF. After each manipula-
tion the resin was washed with DMF. After the last coupling, the
resin was washed consecutively with DMF, DCM and MeOH then
left to dry in vacuo overnight. The peptides were cleaved from the
resin using 95% TFA/2.5% H2O/2.5% TIPS (10–25 mL gꢀ1) for 2 h at
rt. Following this, the slurry was dried, precipitated in diethyl ether,
then dissolved in 1:1 MeCN/H2O and lyophilised.
Compound 1; purified yield: 5.0 mg, 23.3%; HPLC: tR
(C4) = 20.60 min; ESI-MS (C278H402N76O60, 5768.6): m/z = 825.4
[M+7H]+ (calcd 825.1), 962.7 [M+6H]+ (calcd 962.4), 1154.8
[M+5H]+ (calcd 1154.7), 1444.0 [M+4H]+ (calcd 1443.2), 1923.8
[M+3H]+ (calcd 1923.9).
2-Amino-D,L-hexadecanoic acid hydrochloride (2 g, 7.48 mmol)
and Dde-OH (1.5 g, 8.23 mmol) were suspended in ethanol
(30 mL). TEA (2.6 mL) was added and the mixture refluxed under
an inert atmosphere for 2 days. The solvent was evaporated and
the crude product taken up in EtOAc (50 mL) and washed with
5% HCl (3 ꢁ 30 mL) then dried over MgSO4. The solvent was evap-
orated to afford a solid which was triturated with diethyl ether to
afford the pure product as a white solid in 55% yield (1.78 g): Rf
(CHCl3/MeOH, 10:0.7) = 0.58; ESI-MS (C26H45NO4, 435.3):
m/z = 436.4 [M+H]+ (calcd 436.3); 1H NMR: d = 4.38 (1H, dd, J 6.3,
Compound 2; purified yield: 5.8 mg, 26%; HPLC: tR (C4) = 20.63
6.6 Hz,
a
-CH), 2.52 (3H, s, C(NH)CH3), 2.40 (4H, s, 2CH2CO), 2.04–
and 21.47 min; ESI-MS (C284H413N75O64, 5901.8): m/z = 846.4
1.90 (2H, m, b-CH2), 1.46–1.23 (24H, m, 12CH2), 1.02 (6H, s,
C(CH3)2), 0.86 (3H, t, J 6.9 Hz, CH3).
[M+7H]7+ (calcd 844.1), 984.5 [M+6H]6+ (calcd 984.5), 1181.4
[M+5H]5+ (calcd 1181.2).
Compound 3; purified yield: 5.0 mg, 25%; HPLC: tR (C4) = 21.27
4.2.5. Synthesis of lipopeptides
min; tR (C40) = 20.10 min; ESI-MS (C290H426N76O60
, 5936.3):
All peptides were synthesised using standard manual solid
phase peptide synthetic protocols and then purified by RP-HPLC.
Lipopeptides 1-5 were synthesised using p-4-methyl benzhydryl
amine (p-MBHA; substitution 0.45 mmol gꢀ1) resin (Peptides Inter-
national, USA), which was swollen in DMF/DIPEA for approxi-
m/z = 849.4 [M+7H]7+ (calcd 849.0), 990.5 [M+6H]6+ (calcd 990.5),
1188.9 [M+5H]5+ (calcd 1188.3), 1485.1 [M+4H]4+ (calcd 1485.1).
Compound 4; purified yield: 2.0 mg, 56%; HPLC: tR (C4) = 32.34
min; ESI-MS (C179H291N47O44, 3805.51): m/z = 636.4 [M+6H]6+
(calcd 635.2), 763.1 [M+5H]5+ (calcd 762.1), 953.2 [M+4H]4+ (calcd
952.3), 1270.9 [M+3H]3+(calcd 1269.5), 1905.8 [M+2H]2+ (calcd
1903.7).
mately 1 h. Synthesis was carried out using 4 equiv Boc-L-amino
acids. Lipopeptides were synthesised using 8 equiv of activated
amino acids after the addition of the second Lys residue. The pep-
tides were then coupled using amino acids preactivated with an
equimolar amount of 0.5 M HBTU or HATU in DMF and 6 equiv
DIPEA, then mixed with the resin for 30–45 min at rt or for
5–10 min at 70 °C, 20 W. For Boc chemistry synthesis, Boc protect-
Compound 5; purified yield: 8.0 mg, 8%; HPLC: tR (C4) = 22.00,
22.37 min; tR (C40) = 20.90, 22.40 min; ESI-MS (C496H695N139O117,
10475.3): m/z = 953.7 [M+11H]11+ (calcd 953.3), 1049.1
[M+10H]10+ (calcd 1048.5), 1165.2 [M+9H]9+ (calcd 1164.9),
1311.1 [M+8H]8+ (calcd 1310.4), 1498.2 [M+7H]7+ (calcd 1497.5),
1747.7 [M+6H]6+ (calcd 1746.9).
ing groups were used for the
a-amino-termini; Tos for Arg; DNP
and Bom for His; Bzl for Ser; For for Trp; 2-Br-Z for Tyr; 2Cl-Z for
Lys; Xan for Asn and Gln; and OcHx for Glu. Boc-C12-OH and
Boc-C16-OH were used to assemble the N-terminal lipid adjuvant.
Boc-Lys(Boc)-OHꢂDCHA salt was neutralised using 0.5 M NaHSO4/
EtOAc, dried in vacuo and utilised for the synthesis of the polyly-
sine carrier. The Boc protecting groups were removed from the
amino acids using neat TFA. Prior to final Boc deprotection, the
4.2.6. Thioester conjugation of [
adjuvant (6)
D
-Cys6]GnRH to maleimide
The maleimide-T helper peptide adjuvant system, 60 (2.0 mg),
was dissolved in DMF (2 mL). Reduced (by 10 equiv tris(2-carboxy-
ethyl)phosphine, then purified) [
(18 mL) was then added, the reaction degassed, and left to stir at
D
-Cys6]GnRH (2.0 mg) in PBS

D. Goodwin et al. / Bioorg. Med. Chem. 22 (2014) 4848–4854
4853
rt under N2 for 5 h. The product (6) was then purified using RP-
HPLC and characterised with ESI-MS.
and 100 lL of o-phenylenediamine dihydrochloride substrate
(SigmaFast, Sigma–Aldrich) was added. Plates were incubated in
the dark for 30 min and the absorbance of the solutions was then
determined at a wavelength of 450 nm using a Molecular Devices
SpectraMax 250 microplate reader. Cut-off was determined as
the mean of PBS antisera plus 3 standard deviations.
Compound 60; purified yield: 88 mg, 95%; HPLC: tR (C4) =
24.20 min; ESI-MS (C123H211N29O29
,
2559.6): m/z = 1281.0
[M+2H]2+ (calcd 1281.3), 854.3 [M+3H]3+ (calcd 854.9).
Compound 6; purified yield = 2.1 mg, 70%; HPLC: tR
(C4) = 22.32 min; tR (C400) = 15.60, 15.93 min; ESI-MS (C179H288N46O42S,
3788.2): m/z = 758.6 [M+5H]5+ (calcd 758.6), 947.9 [M+4H]4+
(calcd 948.1), 1264.0 [M+3H]3+ (calcd 1263.7), 1895.4 [M+2H]2+
(calcd 1895.1).
4.4.2. Statistics
Statistical analysis was performed using a one-way ANOVA fol-
lowed by the Tukey post hoc test. GraphPad Prism 5 software was
used for statistical analysis, with p <0.05 taken as statistically
significant.
4.3. PBMC isolation and in vitro cell toxicity assay
Assay was undertaken with the approval from the University of
Queensland Ethics Committee (Ethics Approval Number:
2009000661). Blood samples (4 mL) were taken from a healthy
adult consented volunteer and PBMCs were isolated by Ficoll gra-
dient after centrifugation at 400g for 30 min. The buffy coat layer
including mononuclear cells was removed and washed three times
with RPMI 1640 (4 mL). After the last washing step, cells were
resuspended in 10% FBS:RPMI and seeded at 1 ꢁ 106 cells/mL in a
Acknowledgments
We would like to acknowledge the Australian Research Council
(ARC, Australia) for their support of this work with the Discovery
Project Grant (DP110100212), Professorial Research Fellowship to
I.T. (DP110100212) and an Australian Postdoctoral Fellowship to
P.S. (DP1092829). We thank to Mr. Amirreza Rafiee for synthesis
and purification of [
the manuscript.
D
-Cys6]-GnRH and Thalia Guerin for editing
96-well flat bottom plates (TPP), activated by adding 10
of phytohemagglutinin and incubated at 37 °C in a 5% CO2 atmo-
sphere. After 1 h incubation compounds were added at 10 L/well
in at 10, 50, 100 and 200 M. MTT (10 L, 10 mg/mL) (Sigma–
Aldrich) was added to each well and incubated for 4 h. 100 L of
l
g mLꢀ1
l
References and notes
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50 L of PBS. Two boosts (same as the primary dose) were admin-
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din (Sigma–Aldrich) (5
g mlꢀ1) in dH2O at 37 °C on a shaker/
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l
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PBS was added for 1 h at 37 °C. Plates were washed and murine
sera at a 1/100 dilution (continued with a 1:2 dilution down the
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1.5 h at 37 °C in a humidified atmosphere. The sera were removed,
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lL/well) was added and incubated for
l
gated goat anti-mouse IgG (Jackson Laboratories/BioRad, USA) in
1% BSA/PBS was added. After 1 h at 37 °C, the plates were washed

4854
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