Toward the development of prophylactic and therapeutic human papillomavirus type-16 lipopeptide vaccines

Article abstract of DOI:10.1021/jm070287b

Four lipid-core peptide systems were synthesized using stepwise solid-phase peptide synthesis, incorporating a sequence from the human papillomavirus type-16 (HPV-16) E7 protein (E7_44-62), for the purpose of developing vaccines against HPV-16 associated cervical cancer. D-Mannose was conjugated to the vaccine in order to investigate whether the targeting of dendritic cell mannose receptors would improve vaccine efficacy. The ability of the vaccines to clear or reduce the size of HPV-16 associated tumors was assessed in C57BL/6 (H-2^b) mice using the TC-1 HPV-16 tumor model. Overall, significant reductions in the size of TC-1 tumors were observed in the mouse model, with the conjugation of mannose to these vaccines demonstrated to clear or reduce the size of TC-1 tumors to a greater extent than non-mannose-containing vaccines (37 out of 40 versus 21 out of 30 tumors cleared, respectively).

Full text of DOI:10.1021/jm070287b

J. Med. Chem. 2007, 50, 4721-4727
4721
Toward the Development of Prophylactic and Therapeutic Human Papillomavirus Type-16
Lipopeptide Vaccines
†
,#
§
§
§
§
§
#
Peter M. Moyle, Colleen Olive, Mei-Fong Ho, Manisha Pandey, Joanne Dyer, Andreas Suhrbier, Yoshio Fujita, and
Istvan Toth*,
†,#
School of Pharmacy and School of Molecular and Microbial Sciences (SMMS), The UniVersity of Queensland, St. Lucia 4072, Queensland,
Australia, and The Queensland Institute of Medical Research (QIMR), Herston 4029, Queensland, Australia
ReceiVed March 12, 2007
Four lipid-core peptide systems were synthesized using stepwise solid-phase peptide synthesis, incorporating
a sequence from the human papillomavirus type-16 (HPV-16) E7 protein (E744-62), for the purpose of
developing vaccines against HPV-16 associated cervical cancer. D-Mannose was conjugated to the vaccine
in order to investigate whether the targeting of dendritic cell mannose receptors would improve vaccine
efficacy. The ability of the vaccines to clear or reduce the size of HPV-16 associated tumors was assessed
b
in C57BL/6 (H-2 ) mice using the TC-1 HPV-16 tumor model. Overall, significant reductions in the size of
TC-1 tumors were observed in the mouse model, with the conjugation of mannose to these vaccines
demonstrated to clear or reduce the size of TC-1 tumors to a greater extent than non-mannose-containing
vaccines (37 out of 40 versus 21 out of 30 tumors cleared, respectively).
Introduction
HPV vaccines are designed to elicit CD8+ cytotoxic T-
lymphocytes (CTLs) that can eliminate HPV infected cells. The
continued expression of two HPV proteins, E6 and E7, is
required to maintain HPV-associated tumor cell growth. Se-
quences from the E6 and E7 proteins, which contain epitopes
capable of eliciting CTLs, are therefore commonly used for the
development of vaccines against HPV infection.
1
Cervical cancer represents the second most common cancer
worldwide, with 500 000 new cases diagnosed each year,
resulting in 270 000 deaths. Cervical cancer has been identified
as near 100% attributable to human papillomavirus (HPV )
infection of the cervix, with HPV types 16 and 18 responsible
1
a
1
for approximately 50% and 20% of cancers, respectively. The
ability to create prophylactic vaccines targeting these strains
would therefore offer the capacity to prevent up to 70% of
cervical cancers. Vaccines derived from the noninfectious
The current study describes the development of four experi-
mental vaccines (1-4, Table 1) against HPV-16 associated
cervical cancer, synthesized using the lipid-core peptide (LCP)
4
protein coat of HPV-16 and -18, called virus-like particles
system (Table 1). This vaccine delivery system incorporates
1
(
VLPs), have recently been approved for human use. Because
(i) peptides (in this case, HPV peptides designed to elicit HPV-
specific immune responses) attached to a multiple antigen
these vaccines seek to prevent HPV infection, and thus cervical
cancer, they need to be administered prior to the commencement
of sexual activity. A large proportion of the global population
is already infected, and many women will continue to become
infected with cervical cancer-associated HPV strains until global
vaccine coverage is achieved.2 The development of therapeutic
vaccines, capable of eliminating HPV infected cells and clearing
established HPV-associated tumors, would therefore be ben-
eficial. Together, the application of prophylactic and therapeutic
HPV vaccines offers the best approach to achieve HPV
eradication.
5
peptide (MAP) system and (ii) immunopotentiating lipids
6
(lipoamino acids ) to stimulate strong immune responses. The
vaccines contain four copies of a 19 amino acid long sequence
7
(8Q: QAEPDRAHYNIVTFCCKCD; E744-62) from the HPV-
,3
16 E7 protein. This sequence contains a CTL epitope, as well
as a T-helper cell and B-cell epitope. The aim of this study
was to determine whether conjugation of the 8Q peptide to an
LCP system would elicit immune responses capable of protect-
ing against HPV-16 associated tumor development in a mouse
model and whether the attachment of D-mannose to these
vaccines could increase vaccine efficacy, as improvements in
*
To whom correspondence should be addressed. Phone: +61 (7) 3346
8,9
vaccine efficacy have been reported in several systems where
9
892. Fax: +61 (7) 3365 1688. E-mail: i.toth@uq.edu.au.
†
#
antigens have been targeted to mannose receptors.
School of Pharmacy, The University of Queensland.
School of Molecular and Microbial Sciences (SMMS), The University
of Queensland.
Results and Discussion
§
The Queensland Institute of Medical Research (QIMR).
Abbreviations: ANOVA, analysis of variance; BOC, tert-butoxycar-
a
Chemistry. The lipid-core peptide systems 1-7 (Table 1)
and control peptides 8-11 (Table 2) were synthesized using
tert-butoxycarbonyl (Boc) manual stepwise solid-phase peptide
synthesis (SPPS) on p-methylbenzhydrylamine (pMBHA) resin
using 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium
hexafluorophosphate (HBTU)/N,N-diisopropylethylamine (DI-
bonyl; Boc-C12-OH, 2-(tert-butoxycarbonylamino)-D,L-dodecanoic acid;
C12, 2-amino-D,L-dodecanoic acid; CTLs, cytotoxic T-lymphocytes; DCM,
dichloromethane; ddH2O, deionized doubly distilled water; DMF, N,N-
dimethylformamide; DIPEA, N,N-diisopropylethylamine; ESI-MS, electro-
spray ionization mass spectrometry; HBTU, 2-(1H-benzotriazole-1-yl)-
1,1,3,3,-tetramethyluronium hexafluorophosphate; HPLC, high-performance
liquid chromatography; HPV, human papillomavirus; IFA, incomplete
Freund’s adjuvant; IPA, isopropyl alcohol; LCP, lipid-core peptide;
pMBHA, p-methylbenzhydrylamine; MAP, multiple antigen peptide; OVA,
ovalbumin; RP, reversed phase; RT, room temperature; SDS-PAGE,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPPS, solid-
phase peptide synthesis; TCEP, tris(2-carboxyethyl)phosphine hydrochloride;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin-layer chroma-
tography; VLPs, virus-like particles.
10
PEA) in situ neutralization chemistry. The synthesis of LCP
systems 1-7 was achieved by coupling Boc-Gly-OH to the
resin, followed by two cycles of the lipoamino acid 2-(tert-
butoxycarbonylamino)-D,L-dodecanoic acid (Boc-C12-OH), then
Boc-Gly-OH, Boc-C12-OH, Boc-Lys(Boc)-OH, and Boc-Lys-
(Boc)-OH. Deprotection of the N-terminal Boc protecting groups
1
0.1021/jm070287b CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/17/2007

4
722 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Moyle et al.
Table 1. HPV-16 8Q Peptide (1-4) and Control (5-7) LCP Systems
Table 2. Control Peptides
carbohydrate conjugation, a small sample of the resin-bound
mannose 8Q LCP conjugate was kept in order to obtain 3, and
the carbohydrate O-acetyl protection for LCP systems 2 and
4
-6 was removed by treating the resin with 1:11 hydrazine
1
1
hydrate in DMF for 4.5 h. The peptides were subsequently
cleaved from the resins using anhydrous HF, purified by
preparative reversed-phase high-performance liquid chroma-
tography (RP-HPLC), and characterized by analytical RP-HPLC,
electrospray ionization mass spectrometry (ESI-MS, where
possible), and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The synthesis and characteriza-
tion of control peptides 8-11 were performed analogous to the
synthesis and characterization of LCP systems 1-7.
Mannose was attached to LCP systems 2 and 3 in order to
investigate whether targeting of dendritic cell mannose receptors
could increase vaccine efficacy. Improvements in vaccine
efficacy associated with targeting antigens to the mannose
8
,9
receptor have been reported in several systems. As a control,
D-glucose was attached to the 8Q sequence (in the case of LCP
system 4) because it displays weak mannose receptor binding
1
2
in comparison to mannose while maintaining the solubilizing
effects of carbohydrate conjugation.
yielded LCP system 7 on resin. Peptide antigens (Table 1) were
then synthesized onto the four N-terminal amines. For the
synthesis of LCP systems 2-6, the carbohydrates D-mannose
In addition to LCP systems 1-4, controls 5-11 (Tables 1
and 2) were synthesized. The 8Q peptide with (9-11) or without
(
for LCP systems 2, 3, 5, and 6) and D-glucose (LCP system 4)
(
8) attached sugars was synthesized in order to demonstrate
were conjugated to the amino terminus of each peptide antigen.
These carbohydrates were coupled as per-O-acetylated analogues
bearing an N-linked succinate linker (13, 14; Figure 1) and were
synthesized from 2,3,4,6-tetra-O-acetyl protected â-mannopy-
ranosyl (15) and â-glucopyranosylazides by in situ reduction
whether the 8Q peptide on its own, or with attached sugars,
would stimulate appropriate vaccine-mediated immune responses
to clear HPV-16 associated tumors despite not being conjugated
to the LCP system. An LCP core, which contains the MAP
system synthesized without attached peptides (7), and two
mannose-conjugated LCP systems, one (6) containing a ran-
domized version of the 8Q sequence [8Q (random), ADHIFKQ-
PANTCDERYVCC] and another (5) containing a T-helper and
CTL epitope from the ovalbumin (OVA) protein [OVA(Th +
CTL), ISQAVHAAHAEINEAGRSIINFEKL], were synthesized
to demonstrate whether the 8Q HPV peptide is necessary to
protect against HPV-16 associated tumors.
(Pd/C, H2(g)) in the presence of succinic anhydride. Following
Immunological Assessment. Assessment of each vaccine’s
capacity to protect against HPV-16 associated tumor develop-
ment was performed following immunization of C57BL/6 (H-
Figure 1. Acetylated mannose (13) and glucose (14) derivatives.
Carbohydrates 13 and 14 were used for the synthesis of LCP systems
b
13
2
) mice using the TC-1 tumor model. Mice (n ) 10/group)
2
-6, as well as control peptides 9-11.
were immunized subcutaneously at the tail base with each

Prophylactic and HPV-16 Lipopeptide Vaccines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4723
6
Figure 2. The mass of TC-1 tumors excised from mice 15 days after 1 × 10 TC-1 tumor cell challenge in the scruff of the neck (a, b) or lower
back (c, d) following immunization with compounds 1-12 or RPMI 1640 media containing 0.02 mM â-mercaptoethanol. Statistically significant
differences compared with media-immunized mice are represented as follows: (//) P < 0.01; (///) P < 0.001.
immunogen (doses in Tables 1 and 2) administered in RPMI
640 media containing 0.02 mM â-mercaptoethanol. Alterna-
tively, chicken egg OVA and the OVA SIINFEKL CTL epitope,
as well as compound 8, were administered as a 1:1 emulsion in
incomplete Freund’s adjuvant (IFA). Three weeks after im-
with 8Q containing LCP systems (Figure 2b,c). Mice vaccinated
with a mixture of the LCP core (7), which contains the MAP
system synthesized without attached peptides, and mannose
conjugated 8Q (9) developed tumors that did not differ in size
when compared to the RPMI 1640 group (Figure 2c). These
data suggested that LCP systems conjugated to 8Q were capable
of preventing or reducing TC-1 tumor growth in immunized
mice, with mannosylated analogues 2 and 3 appearing to offer
superior anticancer activity and in many cases completely
preventing the formation of detectable tumors (Figure 2).
1
1
3
munization, the mice were challenged with TC-1 tumor cells
(
6
1 × 10 ) either in the scruff of the neck or in the lower back.
These cells express the HPV-16 E6 and E7 proteins, which are
overexpressed in natural HPV-associated cancers. Fifteen days
after TC-1 tumor cell challenge, the tumors were excised and
weighed to assess the effect of immunization on tumor size
To ensure that these results were not due to elicitation of
non-8Q-specific immune responses, mice were immunized with
alternative T-helper and CTL epitopes, the LCP core (7), or an
LCP system incorporating a randomized version of the 8Q
sequence (6). Alternative T-helper and CTL epitopes were
administered as both OVA and the OVA CTL epitope SIIN-
FEKL (12, Table 2) formulated in IFA or as an LCP system
(Figure 2). One-way analysis of variance (ANOVA) was used
to compare the average TC-1 tumor size between groups,
followed by the Bonferroni post hoc test. A P-value of less than
or equal to 0.05 was considered to be significant.
Statistically significant tumor size reductions were observed
in all groups vaccinated with LCP systems 1-3 when compared
with mice that received RPMI 1640 media (Figure 2). Overall,
mice vaccinated with the mannose containing LCP system 2
had the smallest resected tumors (or had no measurable tumors)
(5) incorporating the OVA SIINFEKL CTL epitope and a
T-helper epitope from OVA. In each case (Figure 2a,b) the
average resected tumor sizes were not significantly different
compared to mice given RPMI 1640 media. This demonstrated
that the 8Q peptide sequence was necessary for protection
(Figure 2), followed by mice vaccinated with compound 3
(Figure 2b,d). Mice vaccinated with the non-mannose conjugated
compound 1 showed the largest tumor sizes of mice immunized

4
724 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Moyle et al.
against TC-1 tumor cell challenge and suggests that specific
immune responses were responsible for this activity.
anhydride was purchased from Ajax Finechem (Seven Hills, NSW,
Australia). D-(+)-Mannose was purchased from Lancaster Synthesis
(Lancashire, England). HPLC grade MeCN was purchased from
In order to confirm that the lipid component of the LCP
system was important as an adjuvant, the 8Q peptide (8) was
assessed for its capacity to reduce or prevent TC-1 tumors when
administered without additional adjuvant, either alone or
conjugated to mannose and glucose derivatives (9-11). Mice
vaccinated with compounds 8-11 did not exhibit significant
tumor size reductions compared to mice given RPMI 1640
media. However, it was observed that when some of the
monomeric peptides (8-10) were left in solution for a long
period of time, the larger molecular weight species that formed
as a result of intermolecular disulfide bond formation were able
to stimulate immune responses capable of partially inhibiting
TC-1 tumor growth (unpublished data, PMM & CO). As a
positive control, the 8Q HPV peptide 8 was administered in
IFA, a highly effective adjuvant that is not suitable for human
use. All but 1 of the 40 mice vaccinated with control peptide 8
formulated in IFA were protected against TC-1 tumor cell
challenge (Figure 2).
The 8Q LCP system 1 was observed to be poorly soluble in
RPMI 1640 media and water, while 8Q LCP systems conjugated
to carbohydrates (3 and 4) had greatly improved solubility.
Because mannosylated 8Q LCP systems appear to be more
effective at protecting against TC-1 tumor development com-
pared with the non-mannosylated analogue 1, an attempt was
made to determine whether this may be due to mannose receptor
binding. An experiment (Figure 2d) was performed in which
mice were administered a glucose-containing 8Q LCP system
Labscan (Dublin, Ireland). HPLC grade isopropyl alcohol (IPA)
and MeOH were purchased from Honeywell Burdick and Jackson
(Morristown, NJ). Anhydrous HF was purchased from Matheson
Tri-Gas (Irving, TX). N , H , and Ar gases (all ultrapure grade)
2
2
were purchased from BOC gases (Brisbane, QLD, Australia). The
chicken egg OVA SIINFEKL CTL epitope was purchased from
Mimotopes (Victoria, Australia). All other reagents were purchased
from Sigma-Aldrich (Castle Hill, NSW, Australia) at the highest
available purity. The racemic lipoamino acids 2-amino-D,L-dode-
canoic acid (C12) and Boc-C12-OH were synthesized as previously
described.6
Equipment. An all Kel-F apparatus (Toho Kasei Co., Tokyo,
Japan) was used for HF cleavage. Deionized doubly distilled water
2
(ddH O) was used throughout and was prepared by a Millipore
(Billerica, MA) Simplicity 185 ultrapure water system. Flash
chromatography was performed on silica gel 60 (230-400 mesh;
Lomb scientific, Taren Point, NSW, Australia). Thin-layer chro-
matography (TLC) was performed on Merck (Darmstadt, Germany)
aluminum backed silica gel 60 F254 plates. Detection was achieved
using the following dips with heating for visualization: (a) 20%
(w/v) ninhydrin in EtOH (for amines); (b) p-anisaldehyde dip (135
mL of EtOAc, 5 mL of concentrated H
AcOH, 3.7 mL of p-anisaldehyde); (c) H
2
SO
SO
4
, 1.5 mL of glacial
dip [20% (v/v) H
2
4
2
-
4
SO in EtOH]. Uncorrected melting points (°C) were obtained on
a Buchi (Switzerland) melting point apparatus. Optical rotation was
measured at room temperature (RT) in a 10 cm cell using a Perkin-
1
13
Elmer (Wellesley, MA) 241MC polarimeter. H and C NMR
spectra were recorded on a Bruker Avance 500 MHz spectrometer
(Bruker Biospin, Germany) at 298 K in deuterated chloroform
(4) as well as mannose-containing 8Q LCP systems 2 and 3
(CDCl ; Cambridge Isotope Laboratories, Andover, MA). Chemical
3
and LCP system 1. The results suggested that the glucose-
containing LCP system 4 was not as effective at reducing the
size of, or preventing, TC-1 tumors compared with LCP systems
shifts are reported in parts per million (ppm) downfield from
tetramethylsilane, with chemical shifts referenced to the residual
CHCl peak (δ 7.24 ppm). NMR spectra were processed using
3
Topspin 1.3 (Bruker Biospin, Germany). Elemental analysis was
performed on a Carlo Erba (Lakewood, NJ) elemental analyzer
model 1106. SDS-PAGE was performed using precast 12% Bis-
Tris Novex gels (Invitrogen), and Multimark colored standards
1
-3. However, there were no statistically significant differences
in tumor sizes between these groups.
Conclusion
(Invitrogen, 10 µL), in a Novex Xcell Surelock Mini-Cell (Invit-
Conjugation of mannose residues to 8Q containing LCP
systems, both in per-O-acetylated and nonacetylated forms,
resulted in smaller tumor sizes compared with non-carbohydrate
conjugated and glucose conjugated analogues. Furthermore,
incorporation of the 8Q peptide into the LCP system was
observed to be essential for protection against TC-1 tumor cell
challenge, since incorporation of a randomized 8Q sequence or
alternative CTL and T-helper epitopes did not significantly affect
tumor size when compared to media-immunized mice. The LCP
systems incorporating the 8Q peptide (1-4) were also demon-
strated to be self-adjuvanting and elicited vaccine-mediated
immune responses capable of preventing TC-1 tumors, with the
rogen). The gels were subsequently stained using SimplyBlue
SafeStain (Invitrogen, 20 mL) and imaged using a flat-bed scanner
(Canon Canoscan N640P).
Analytical RP-HPLC was performed using Shimadzu (Kyoto,
Japan) instrumentation (Class Vp 6.12 software, SCL-10AVp
controller, SIL-10A autoinjector, LC-10AT pump, LC-10AD pump,
Waters 486 tunable absorbance detector). Analytical RP-HPLC was
performed in gradient mode using a 1 mL/min flow rate, with
detection at 214 nm. For all separations, except for peptide 10,
solvent A consisted of 0.1% (v/v) aqueous TFA and solvent B
consisted of either 90% MeCN/0.1% TFA/H
IPA/0.1% TFA/H O (solvent B2), or 90% MeOH/0.1% TFA/H
solvent B3). For peptide 10, solvent A consisted of H O acidified
2
O (solvent B1), 90%
2
2
O
(
2
8
Q peptide established to be incapable of preventing TC-1
to pH 6 (AcOH) and solvent B consisted of 90% MeCN/H O
2
tumors when delivered on its own, either unconjugated or
conjugated to mannose or glucose residues or physically mixed
with the LCP core 7. In conclusion, the current study has
demonstrated the capacity of the LCP system to adjuvant the
CTL epitope-containing 8Q HPV-16 E7 peptide in preventing
HPV-16 associated tumors and its potential for the development
of prophylactic and possibly therapeutic human lipopeptide HPV
vaccines.
acidified to pH 6 (AcOH). Separation was achieved on a Vydac
(Hesperia, CA) analytical C18 column (C18, 218TP54, 5 µm, 4.6
mm × 250 mm), a Vydac analytical C4 column (C4, 214TP54, 5
µm, 4.6 mm × 250 mm), or a short Vydac analytical C4 column
(
C4s, 214TP5405, 5 µm, 50 mm × 4.6 mm). All gradients were
run over 30 min.
Preparative-scale RP-HPLC was performed on a Waters Delta
600 system (600E solvent delivery module; 490E multiwavelength
UV detector; Fraction Collector III). Preparative RP-HPLC was
performed in gradient mode using a 10 mL/min flow rate (unless
otherwise specified) with detection at 230 nm. For all separations,
except for peptides 3 and 10, solvent A consisted of 0.1% (v/v)
aqueous TFA and solvent B consisted of 90% MeCN/0.1% TFA/
Experimental Section
Materials and Methods. Boc-L-amino acids and pMBHA resin
were purchased from Renanal (Budapest, Hungary). Peptide
synthesis grade N,N-dimethylformamide (DMF), dichloromethane
2 2
H O. For peptides 3 and 10 solvent A consisted of H O acidified
(
DCM), HBTU, and trifluoroacetic acid (TFA) were purchased from
to pH 6 with AcOH, and solvent B consisted of 90% (v/v) aqueous
MeCN acidified to pH 6 with AcOH. The following columns were
Auspep (Melbourne, VIC, Australia). Analytical grade acetic

Prophylactic and HPV-16 Lipopeptide Vaccines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4725
utilized for separation: Vydac C4 preparative column (C4,
4-(2,3,4,6-Tetra-O-acetyl-â-D-glucopyranosylamino)-4-oxobu-
tanoic Acid (14). Succinic anhydride (1.09 g; 10.9 mmol) and
2,3,4,6-tetra-O-acetyl-â-D-glucopyranosylazide16 (3.7 g, 9.9 mmol)
were dissolved in dry THF (40 mL). This solution was treated with
a crystal of 4-di(methylamino)pyridine and 10% Pd/C (200 mg),
degassed, and left to stir at RT under H2(g) for 24 h. The solution
was then concentrated, taken up in EtOAc (150 mL), and filtered
through Celite. The filtrate was washed with 5% (v/v) HCl (3 ×
2
14TP1022, 10 µm, 250 mm × 22 mm), Vydac C18 preparative
column (C18, 218TP1022, 10 µm, 250 mm × 22 mm), or a Vydac
C4 semipreparative column (C4SP, 214TP1010, 10 µm, 250 mm
×
10 mm). Prior to purification of the cysteine containing peptides,
cysteine residues were reduced by treating each peptide with tris-
2-carboxyethyl)phosphine hydrochloride (TCEP, approximately 3
(
equiv per cysteine residue).
3
0 mL) and saturated NaCl solution (3 × 30 mL), dried (MgSO
4
),
ESI-MS was performed on a Perkin-Elmer-Sciex API3000 triple
quadrupole mass spectrometer fitted with an atmospheric pressure
ESI source. The ion spray source was coupled to a binary Shimadzu
HPLC system (SCL-10Avp system controller, 2 × LC-10ATvp
pumps, DGU-12A degasser unit, Agilent 1100 series standard
autosampler). Samples (1-10 µL) were injected via a manual
and concentrated to give white crystals, which were triturated with
hexane, filtered, and dried to give 14 (3.15 g, 71.3% yield) as white
crystals. TLC R
55 °C. Anal. (C18
NHCO(CH ) COOH
f
) 0.38 (CHCl
25NO12) C, H, N. ESI-MS: m/z [M -
331.4 (calcd 331.3), [M + H 448.4
3
/MeOH/AcOH, 90:8:2); mp 152-
1
H
]
-
+
+ +
2
2
]
+
+
1
(calcd 448.4), [M + Na ] 470.3 (calcd 470.4). H NMR (500
2
injection port (Rheodyne, CA) into MeCN-H O mobile phases
MHz, CDCl ): δ 1.99, 2.01, 2.03, 2.05 (4 × 3H, s, CH ), 2.46
3
3
containing 0.1% (v/v) formic acid. ESI-MS data was acquired using
Analyst 1.4 (Applied Biosystems/MDS Sciex, Toronto, Canada)
software. All samples were run in positive ion mode.
2
(2H, m, CH ), 2.61 (1H, m, CH
a
), 2.72 (1H, m, CH
b
), 3.81 (1H,
m, H-5), 4.06 (1H, dd, J 12.7, 2.0 Hz, H-6a), 4.27 (1H, dd, J 12.7,
4.0 Hz, H-6b), 4.91 (1H, t, J 9.5 Hz, H-2), 5.04 (1H, d, J 9.9 Hz,
H-4), 5.23 (1H, d, J 9.0 Hz, H-1), 5.28 (1H, t, J 9.5 Hz, H-3), 6.57
Carbohydrate Synthesis. 2,3,4,6-Tetra-O-acetyl-â-D-man-
nopyranosylazide (15). Sodium azide (10.7 g; 170 mmol) was
added to 2,3,4,6-tetra-O-acetyl-R-D-mannopyranosyl bromide14
(
1H, d, J 9.5 Hz, NH). 13C NMR (125 MHz, CDCl
3
): δ 20.55,
2
6
1
0.55, 20.58, 20.70 (4 × CH
3
), 28.59, 30.64 (2 × CH
2
), 61.66 (C-
), 68.12 (C-4), 70.57 (C-2), 72.72 (C-3), 73.55 (C-5), 78.14 (C-
), 169.60, 169,92, 170.72, 171.25, 171.96, 176.36 (6 × CdO).
Peptide Synthesis. Peptides were synthesized by manual step-
(33.8 g, 82.2 mmol) dissolved in anhydrous DMF (140 mL) and
left to stir at 70 °C under Ar(g). After 13 h the mixture was
concentrated, dissolved in EtOAc (200 mL), and filtered to remove
wise SPPS on pMBHA resin using HBTU/DIPEA in situ neutral-
inorganic matter. The filtrate was washed with saturated NaHCO
solution (3 × 50 mL), 5% (v/v) HCl (3 × 50 mL), and saturated
NaCl solution (50 mL), dried (MgSO ), filtered, and concentrated
to give a light-brown oil. The product was crystallized from EtOH
3
10
ization and Boc chemistry. Prior to peptide synthesis, the resin
was swollen in anhydrous DMF (10 mL/g resin) for 1 h. The HCl
salt was then neutralized by 3 × 15 min treatments with 10% (v/v)
DIPEA in anhydrous DMF (10 mL/g of resin) prior to peptide
synthesis. Each amino acid coupling cycle consisted of Boc
deprotection (2 × 1 min treatments with neat TFA), a 1 min DMF
flow-wash, followed by 15-60 min couplings with 4 equiv of
preactivated amino acid. Coupling yields were determined using
4
(
R
[
30 mL) to afford 15 (4.18 g, 13.6% yield) as white crystals. TLC
15
f
) 0.79 (EtOAc); mp 114-116 °C (literature value 124 °C );
25
15
R] -76.4° (c 0.965, CHCl
3
) (literature value -77°, CHCl
3
).
D
+
+ +
ESI-MS: m/z [M - N
(
CDCl
3
] 331.19 (calcd 331.3), [M + Na ] 396.10
1
17
calcd 396.3). Anal. (C14
H
19
N
O
3 9
) C, H, N. H NMR (500 MHz,
), 3.74 (1H, m,
the quantitative ninhydrin test. Where necessary, couplings were
repeated to give coupling yields greater than 99.7%. For coupling
3
): δ 1.97, 2.03, 2.09, 2.19 (4 × 3H, s, CH
3
to a proline residue, the chloranil test1
8,19
was utilized instead of
H-5), 4.19 (1H, dd, J 12.5, 2.4 Hz, H-6a), 4.26 (1H, dd, J 12.3, 5.6
Hz, H-6b), 4.70 (1H, d, J 1.0 Hz, H-1), 5.02 (1H, dd, J 10, 3.2 Hz,
the ninhydrin test. Amino acid activation was achieved by dissolving
amino acids (4.4 equiv) in 0.5 M HBTU/DMF solution (4 equiv)
to which DIPEA (12 equiv) was added. Amino acids were
preactivated for 1 min prior to their addition to the resin. The same
activation method was utilized for sugar derivatives 13 and 14 (with
H-3), 5.24 (1H, t, J 10 Hz, H-4), 5.43 (1H, d, J 2.2 Hz, H-2). 13
C
NMR (125 MHz, CDCl
6
8
3
): δ 20.47, 20.60, 20.66, 20.66 (4 × CH
2.29 (C-6), 65.39 (C-4), 69.24 (C-2), 70.95 (C-3), 74.67 (C-5),
5.12 (C-1), 169.49, 169.88, 169.88, 170.55 (4 × CdO).
-(2,3,4,6-Tetra-O-acetyl-â-D-mannopyranosylamino)-4-ox-
obutanoic Acid (13). Succinic anhydride (0.5 g, 5.0 mmol) and
3
),
5
.74 equiv of DIPEA). Boc-amino acids with the following side
chain protection were utilized for peptide synthesis: Arg(Tos), Asn-
Xan), Asp(OcHx), Cys(pMeBzl), Gln(Xan), Glu(OcHx), His-
4
(
15 (1.0 g, 2.7 mmol) were dissolved in dry tetrahydrofuran (THF,
(DNP), Lys(2-Cl-Z), Ser(Bzl), Thr(Bzl), Tyr(2-Br-Z). The synthetic
6
25 mL). This solution was treated with 10% Pd/C (100 mg),
racemic lipoamino acid Boc-C12-OH was utilized for LCP lipid
degassed, and left to stir at RT under H2(g) for 2 days. The solution
was concentrated, taken up in EtOAc (150 mL), and filtered through
Celite. The filtrate was washed with 5% (v/v) HCl (3 × 50 mL),
core synthesis. After coupling glutamine residues, the resin was
washed with DCM before and after Boc deprotection to prevent
high-temperature catalyzed pyrrolidone carboxylic acid formation.
Acetylation was achieved by treating the resin with a mixture of
acetic anhydride (0.5 mL), DIPEA (0.47 mL), and DMF (14 mL)
for 5 min and repeating for 30 min. Following peptide synthesis,
the following procedures were performed prior to peptide cleavage
where necessary: (1) For peptides containing His(DNP) residues
10
4
dried (MgSO ), and concentrated to give an orange oil. The oil
was dissolved in a minimum volume of 9:1:0.1 EtOAc/hexane/
AcOH, applied to a column (18 cm × 2.5 cm) of silica gel 60, and
eluted with 9:1:0.1 EtOAc/hexane/AcOH. The fractions containing
the pure product, as confirmed by TLC and ESI-MS, were
combined, concentrated, dissolved in toluene (30 mL), and con-
(1-6, 8-11), the DNP group was cleaved by treating the resin
with 20% (v/v) â-mercaptoethanol, 10% (v/v) DIPEA in DMF for
2 × 1 h treatments. (2) For peptides containing an N-terminal Boc-
protecting group (1, 7, 8), the Boc-protecting group was removed,
followed by a 1 min DMF flow-wash. (3) Peptides 2, 4-6, 9, and
centrated to give 13 (0.38 g, 32% yield) as a white foam. TLC R
f
25
)
0.3 (9:1:0.1 EtOAc/hexane/AcOH); [R]D -11.0° (c 0.807,
CHCl
3
). Anal. (C18
H
25NO12) C, H, N. ESI-MS: m/z [M -
-
+
+ +
NHCO(CH
2
)
2
COOH ] 331.21 (calcd 331.3), [M + H ] 448.32
1
1 were de-O-acetylated by agitating the resin in 1:11 hydrazine
+
+
1
(
calcd 448.4), [M + Na ] 470.33 (calcd 470.4). H NMR (500
MHz, CDCl ), 2.49,
): δ 1.95, 2.02, 2.06, 2.20 (4 × 3H, s, CH
.66 (2 × 2H, m, -CH -CH -), 3.76 (1H, m, H-5), 4.07 (1H,
dd, J 12.4, 2.1 Hz, H-6a), 4.27 (1H, dd, J 12.4, 5 Hz, H-6b), 5.10
1H, dd, J 10, 3.3 Hz, H-3), 5.19 (1H, t, J 10 Hz, H-4), 5.32 (1H,
t, J 3.1 Hz, H-2), 5.52 (1H, d, J 9.3 Hz, H-1), 6.66 (1H, d, J 9.3
Hz, -NH-). 13C NMR (125 MHz, CDCl
): δ 20.82, 20.87, 20.95,
1.03 (4 × CH ), 29.16 (CH ), 30.95 (CH ), 62.48 (C-6), 65.51
C-4), 70.21 (C-2), 71.80 (C-3), 74.34 (C-5), 76.34 (C-1), 169.67,
69.93, 170.68, 170.84, 171.27, 176.14 (6 × CdO).
11
hydrate in DMF for 4.5 h. Following removal of these protecting
groups, the resin was thoroughly washed with DMF, DCM, and
MeOH and dried (vacuum desiccator) prior to cleavage using
anhydrous HF.
3
3
2
2
2
(
HF Cleavage. HF cleavage (10 mL of HF/g of resin) was
performed at -5 °C for 2 h. p-Cresol (5% (v/v)) was utilized as a
scavenger, with 5% (v/v) p-thiocresol also added where peptides
contained pMeBzl protected Cys residues (1, 2, 4, 6, 8, 9, 11). For
peptides containing O-acetyl protected sugars (3, 10), 10% (v/v)
p-cresol was used as a scavenger, with no p-thiocresol added.
3
2
3
2
2
(
1

4
726 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Moyle et al.
20.925 and 21.208 min (0-100% solvent B2, C4 column), t )
Following HF cleavage, the HF was removed under reduced
pressure. The peptides were then precipitated in ice-cold ether,
filtered, and washed with ice-cold ether. The peptides, except for
R
27.933 min (20-100% solvent B3, C4 column). HPLC purity,
+
2+
g95%. ESI-MS: m/z [M + H] 1109.1 (calcd 1108.6), [M + 2H]
3
and 10, were then dissolved in 40% MeCN/0.1% TFA/H
2
O,
555.4 (calcd 554.8). MW 1107.60 g/mol.
frozen using MeOH-dry ice, and lyophilized to give the crude
Analytical Data for Control Peptide 8. Yield: 6.5%. HPLC:
) 16.125 min (0-100% solvent B1, C18 column), t ) 21.792
min (10-50% solvent B1, C18 column), t ) 14.258 min (0-
100% solvent B2, C18 column), t ) 24.225 min (0-100% solvent
B3, C18 column). HPLC purity ) 95.3%. ESI-MS: m/z [M +
peptides. Alternatively, peptides 3 and 10 were dissolved in 50%
t
R
R
(
v/v) aqueous MeCN, frozen using MeOH-dry ice, and lyophilized
R
to give the crude peptides.
R
Synthesis of LCP Systems 1-7. Synthesis of LCP systems 1-7
was performed as previously described.4 Briefly, Boc-Gly-OH
was coupled to pMBHA resin, followed by two cycles of Boc-
C12-OH, then Boc-Gly-OH, Boc-C12-OH, Boc-Lys(Boc)-OH, and
Boc-Lys(Boc)-OH. Following deprotection of the N-terminal Boc
protecting groups, a portion of the resin was washed, dried, and
cleaved to yield 7. For peptides 1-6, four copies of the peptide
epitope of interest were synthesized on the exposed amines. The
N-terminal Boc protecting groups were then removed. For peptide
,20
+ 2+
+ 3+
2H ] 1107.8 (calcd 1107.2), [M + 3H ] 739.0 (calcd 738.5),
+
4+
+ 6+
[M + 4H ] 554.8 (calcd 554.1), [M + 6H ] 371.5 (calcd
369.7). MW 2212.49 g/mol.
Analytical Data for Control Peptide 9. Yield: 7.5%. HPLC:
t
R
) 16.025 min (0-100% solvent B1, C18 column), t
min (10-50% solvent B1, C18 column), t ) 14.008 min (0-
100% solvent B2, C18 column), t ) 23.708 min (0-100% solvent
R
) 21.567
R
R
B3, C18 column). HPLC purity ) 98.4%. ESI-MS: m/z [M +
+
2+
+ 3+
1, the resin was then washed, dried and cleaved. For peptides 2-6,
2H ] 1238.2 (calcd 1237.9), [M + 3H ] 825.6 (calcd 825.6).
the modified carbohydrate of interest (13 or 14) was coupled to
the N-terminal amines. Peptides 2, and 4-6 were then de-O-
acetylated. The resins were then washed, dried, and cleaved to yield
the crude peptides, which were semipurified by preparative RP-
HPLC.
MW 2473.72 g/mol.
Analytical Data for Control Peptide 10. Yield: 18.6%.
HPLC: t
solvent B, C4 column), t
min (0-100% solvent B, C4s column), t
9.317 (-1Ac) min (0-100% solvent B2, C4s column). HPLC purity
R
) 15.833 (-2Ac) and 15.492 (-1Ac) min (0-100%
) 11.183 (-2Ac) and 11.458 (-1Ac)
) 9.042 (-2Ac) and
R
R
Analytical Data for LCP System 1. Purified yield: 51.5%.
2+
HPLC: t
R
) 21.292 min (0-100% solvent B1, C4 column), t
R
)
) N/A. ESI-MS: m/z [M - Ac + 2H] 1301.1 (calcd 1300.0),
[M - 2Ac + 2H]2 1280.6 (calcd 1279.0), [M - Ac + 3H]
+
3+
1
1
8.858 min (0-100% solvent B2, C4 column). SDS-PAGE: MW
+
7+
3+
1 kDa (expected 9.9 kDa). ESI-MS: m/z [M + 7H ] 1412.4
868.4 (calcd 867.0), [M - 2Ac + 3H] 854.2 (calcd 853.0). MW
(-Ac) 2598.08 g/mol. MW (-2Ac) 2556.07 g/mol.
Analytical Data for Control Peptide 11. Yield: 12.9%.
+
9+
+ 12+
(calcd 1413.7), [M + 9H ] 1100.4 (calcd 1099.8), [M + 12H ]
8
24.6 (calcd 825.1). MW 9889.45 g/mol.
Analytical Data for LCP System 2. Purified yield: 45.2%.
HPLC: t
R
) 15.842 min (0-100% solvent B1, C18 column), t )
R
HPLC: t
R
) 21.833 min (0-100% solvent B1, C4 column), t
R
)
18.458 min (10-60% solvent B1, C18 column), t ) 14.508 min
R
1
1
(
1
[
8.192 min (0-100% solvent B2, C4 column). SDS-PAGE: MW
(0-100% solvent B2, C18 column), 23.950 min (0-100% solvent
+
8+
+ +
2 kDa (expected 10.9 kDa). ESI-MS: m/z [M + 8H ] 1367.3
B3, C18 column). HPLC purity, g95%. ESI-MS: m/z [M + H ]
+
10+
+ 2+
calcd 1367.8), [M + 10H ]
1094.5 (calcd 1094.4), [M +
2474.7 (calcd 2474.7), [M + 2H ] 1238.5 (calcd 1237.9), [M +
+
11+
+ 12+
+ 3+
1H ] 994.9 (calcd 995.0), [M + 12H ] 913.2 (calcd 912.2),
3H ] 826.6 (calcd 825.6). MW 2473.72 g/mol.
+
14+
+ 15+
M + 14H ] 781.3 (calcd 782.0), [M + 15H ] 729.8 (calcd
Immunological Assessment of LCP Systems (1-7) and
Control Peptides (8-12). All protocols were approved by the
Bancroft Centre Research Animal Ethics Committee (approval
+
16+
+ 17+
7
(
29.9), [M + 16H ] 683.4 (calcd 684.4), [M + 17H ] 645.3
+
18+
calcd 644.2), [M + 18H ] 608.3 (calcd 608.5). MW 10 934.36
g/mol.
Analytical Data for LCP System 3. Purified yield: 45.4%.
HPLC: t ) 21.133 min (0-100% solvent B1, C4 column), t
9.033 min (0-100% solvent B2, C4 column). SDS-PAGE: MW
2 kDa (expected 11.6 kDa); MW 11606.95 g/mol. Treating 5 mg
b
number P415). C57BL/6 (H-2 ) mice (10/group) were immunized
at the tail base with compounds 1-7 and 9-11 in RPMI 1640
media, with 8 in RPMI 1640 media or emulsified in incomplete
Freund’s adjuvant (IFA), and with OVA/SIINFEKL (12) in IFA.
For priming, 30 µg of immunogen [40 µg for carbohydrate
containing immunogens (2-6, 9-11); 1µg/10µg for the OVA/
SIINFEKL group (12)] was administered in either IFA (1:1) or
RPMI media (50 µL total volume). â-Mercaptoethanol (0.02 mM)
was included in all samples to prevent cysteine oxidation prior to
immunization. Three weeks after immunization, the mice were
challenged with TC-1 cells13 (1 × 10 ) injected into the scruff of
the neck (parts a and b of Figure 2) or lower back (parts c and d
of Figure 2). Fifteen days after the tumor cell challenge, tumors
were excised and weighed to investigate tumor growth.
R
R
)
1
1
of 3 with 0.6 M aqueous LiOH (degassed with He(g)) for 2 h at RT
to cleave O-acetyl protection) followed by acidification and
reduction with TCEP (approximately 3 eq/cys) yielded an ESI-MS
spectrum identical to that for LCP system 2.
(
Analytical Data for LCP System 4. Purified yield: 10.6%.
6
HPLC: t
R
) 20.467 min (0-100% solvent B1, C4 column), t
R
)
1
1
(
7
1
7.342 min (0-100% solvent B2, C4 column). SDS-PAGE: MW
+
10+
2 kDa (expected 10.9 kDa). ESI-MS: m/z [M + 10H ] 1095.1
+
11+
+ 14+
calcd 1094.4), [M + 11H ] 994.3 (calcd 995.0), [M + 14H ]
+
15+
82.4 (calcd 782.0), [M + 15H ]
729.3 (calcd 729.9), [M +
684.1 (calcd 684.4). MW 10 934.36 g/mol.
Analytical Data for LCP System 5. Purified yield: 69.0%.
HPLC: t ) 21.925 min (0-100% solvent B1, C4 column), t
0.100 min (0-100% solvent B2, C4 column). SDS-PAGE: MW
Statistical Analysis. Comparison of the average resected TC-1
tumor masses between groups was performed using a one-way
ANOVA followed by the Bonferroni post hoc test. GraphPad Prism
4 was used for statistical analysis, with P < 0.05 taken as
statistically significant.
+
16+
6H ]
R
R
)
2
1
(
1
1
+
9+
2 kDa (expected 12.9 kDa). ESI-MS: m/z [M + 9H ] 1441.3
+
10+
Acknowledgment. P.M.M. and C.O. equally contributed to
this work and are joint first authors. C.O. is an R. Douglas
Wright Research Fellow (National Health and Medical Research
Council of Australia). P.M.M. acknowledges the financial
support of the Queensland Government through the award of a
Growing the Smart State Ph.D. funding scholarship. TC-1 cells
were kindly provided by Professor Ian Frazer (Centre for
Immunology & Cancer Research, University of Queensland).
calcd 1440.6), [M + 10H ]
1295.7 (calcd 1296.7), [M +
+
11+
+ 12+
1H ]
1177.3 (calcd 1178.9), [M + 12H ]
1079.7 (calcd
+
15+
080.7), [M + 15H ] 865.4 (calcd 865.4). MW 12 956.54 g/mol.
Analytical Data for LCP System 6. Purified yield: 58.4%.
HPLC: t
R
) 19.842 min (0-100% solvent B1, C4 column), t )
R
1
8.208 (0-100% solvent B2, C4 column). SDS-PAGE: MW 11
+
10+
kDa (expected 10.9 kDa). ESI-MS: m/z [M + 10H ]
1094.5
+
11+
+ 12+
(
9
1
[
calcd 1094.4), [M + 11H ] 994.9 (calcd 995.0), [M + 12H ]
+
14+
13.0 (calcd 912.2), [M + 14H ]
781.3 (calcd 782.0), [M +
+
15+
+ 16+
5H ] 729.7 (calcd 729.9), [M + 16H ] 683.7 (calcd 684.4),
Supporting Information Available: Elemental analysis results,
SPPS details, preparative RP-HPLC data, and SDS-PAGE details.
This material is available free of charge via the Internet at http://
pubs.acs.org.
+
17+
M + 17H ]
Analytical Data for LCP System 7. Yield: 6.9%. HPLC: t
22.658 and 23.033 min (0-100% solvent B1, C4 column), t
645.3 (calcd 644.2). MW 10r t934.36 g/mol.
R
)
R
)

Prophylactic and HPV-16 Lipopeptide Vaccines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4727
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