Development of a liposaccharide-based delivery system and its application to the design of group A streptococcal vaccines

Article abstract of DOI:10.1021/jm701410p

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of D-glucose and D-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the D-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2^k) mice with the liposaccharide vaccine candidates.

Full text of DOI:10.1021/jm701410p

J. Med. Chem. 2008, 51, 1447–1452
1447
Development of a Liposaccharide-Based Delivery System and Its Application to the Design of
Group A Streptococcal Vaccines
Pavla Simerska,† Abu-Baker M. Abdel-Aal,† Yoshio Fujita,† Peter M. Moyle,† Ross P. McGeary,† Michael R. Batzloff,‡
Colleen Olive,‡ Michael F. Good,‡ and Istvan Toth*,†
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 NoVember 8, 2007
Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to
life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates
as carriers for peptide antigens. This study describes the optimized synthesis of D-glucose and D-galactose
derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups.
Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M
protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain
peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to
70% of GAS strains. Lipophilic amino acids were also conjugated to the D-glucose anomeric carbon to
produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the
incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2k) mice
with the liposaccharide vaccine candidates.
Introduction
The coupling of lipids to peptide antigens may be performed
via an amplifying dendrimer carrier system such as the multiple
antigen peptide (MAP) system,10 which polymerizes peptide
antigens through a branched polylysine core. In animal studies,
the immune responses elicited against a peptide antigen were
increased when the antigen was conjugated to a MAP system
compared to when the peptide antigen was administered on its
own.11 Recent studies12–15 have investigated vaccine develop-
ment using the lipid core peptide (LCPa) system, a vaccine
delivery system that conjugates lipoamino acids (R-amino acids
with long alkyl side chains) through a MAP system to multiple
copies of one or more peptide antigens. The LCP system
provides similar immune responses to antigens coadministered
with conventional adjuvants and represents a promising system
for mucosal vaccine development.16
The advent of new, efficient, and sensitive analytical and
synthetic methods in carbohydrate chemistry, together with an
increased understanding of glycobiology and glycoimmunology,
makes the development of carbohydrate-based vaccines possible.
Carbohydrates, as carriers, similarly to the MAP system,13
provide numerous attachment points for the conjugation of one
or more copies of same or different peptide antigens. Further-
more, by using different carbohydrates, the stereochemistry of
the vaccine may be altered to provide an optimal orientation
for the recognition of peptide epitopes by cells of the immune
system. Carbohydrate carriers also help to reduce degradation
of the attached peptide antigens. Therefore, the conjugation of
peptide antigens to lipids (as an adjuvant) and carbohydrates
(as a carrier) represents a highly promising strategy for the
development of peptide vaccines.
Vaccines are considered to be one of the safest and most cost-
effective public health interventions, preventing up to three
million deaths each year.1 Problems with conventional vaccines
including instability (necessitating their transport and storage
under cold chain conditions), the paucity of carriers and
adjuvants suitable for human use, and the potential dangers of
using live microorganisms (risk of infection/reversion to
virulence, allergies, and autoimmune responses) have led to the
development of synthetic vaccines.2 Peptide vaccines have many
advantages over conventional vaccines. For example, peptide
vaccines may incorporate only the minimal microbial compo-
nents required to elicit an effective immune response, thereby
reducing the risk of side effects and autoimmune responses
associated with the administration of unnecessary microbial
components. The development of synthetic peptide vaccines still
faces many hurdles, however (e.g., toxicities associated with
the administration of adjuvants). Although many experimental
adjuvants are available, very few (e.g., squalene oil–water
emulsions, calcium- and aluminum-based salts, cholera toxin
B subunit, and monophosphoryl lipid A) have been used in
vaccines approved for human administration.3 This lack of
suitable adjuvants for human use has spawned a need to develop
new adjuvants. One such approach involves the conjugation of
lipids to peptides to impart adjuvant activity upon the conjugated
peptide antigens. These lipopeptide vaccines may constitute an
ideal strategy for producing vaccines against pathogens that
infect mucosal surfaces and may hold potential for the delivery
of nonimmunogenic peptides and drugs orally.4,5 Lipopeptide
vaccine approaches have been studied for the development of
vaccines against human immunodeficiency virus type-1 (HIV-
1),6 hepatitis B7 and C viruses,8 and malaria.9
Group A streptococcal infection is caused by the bacterium
Streptococcus pyogenes and may cause a variety of health
problems ranging from a sore throat (e.g., Streptococcal
a Abbreviations: Boc, t-butoxycarbonyl; CFA, complete Freund’s adju-
vant; GAS, group A streptococcus; LCP, lipid core peptide; PBS, phosphate-
buffered saline; pMBHA, p-methylbenzhydrylamine; RF, rheumatic fever;
RHD, rheumatic heart disease.
* To whom correspondence should be addressed. Phone: +61 (7) 3346
9892. Fax: +61 (7) 3365 4273. E-mail: i.toth@uq.edu.au.
†
The University of Queensland.
‡
The Queensland Institute of Medical Research.
10.1021/jm701410p CCC: $40.75
2008 American Chemical Society
Published on Web 02/16/2008

1448 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Simerska et al.
Scheme 1. Synthesis of the Glucose/Galactose-Lipid Core Templates (6a, 6b, and 6c) Bearing R ) J8 or J14 Peptide Antigens,
Respectivelya
a Reagents and conditions: (i) acrylonitrile, DBU, ACN; (ii) H2/Pd-C, THF; HBTU, DIPEA, THF, monobenzyl adipate; (iii) NaBH4, CoCl2 ·6H2O, Boc2O,
MeOH; H2/Pd-C; (iv) C12-G-C12-C12-G-NH-MBHA resin; (v) stepwise solid-phase peptide synthesis of J8 (QAEDK VKQSR EAKKQ VEKAL KQLED
KVQ) or J14 (KQAEDK VKASR EAKKQ VEKAL EQLED KVK); HF cleavage.
pharyngitis) to the potentially fatal poststreptococcal sequelae
rheumatic fever (RF) and rheumatic heart disease (RHD).
Together, RF and RHD are responsible for more than 517000
deaths per annum (World Health Organization report).17,18 The
GAS cell-surface M protein, of which over 150 different types
have been identified,19 is considered to be an important target
for the development of prophylactic GAS vaccines. Current
vaccination approaches include the use of multiepitope vaccines
incorporating epitopes derived from the highly variable M
protein amino-terminus and/or the M protein carboxy-terminal
region,18,20,21 which is more highly conserved between GAS
strains.
The current study describes the design of a synthetic subunit
vaccine delivery system incorporating a carbohydrate template,
which acts as a carrier for multiple copies of the J8 or J14
antigens; chimeric peptides derived from the carboxy-terminal
C-repeat region of the GAS coiled-coil R-helical surface M
protein (peptide 145).22 This glycopeptide in turn was conjugated
to a lipid moiety, synthesized using lipidic amino acids, to impart
adjuvant activity upon the vaccine construct. To optimize the
delivery system, different carbohydrate entities (D-glucose and
D-galactose) were synthesized and characterized prior to their
incorporation into lipid-carbohydrate-peptide (lipoglycopep-
tide) vaccines. Subcutaneous immunization of B10.BR (H-2k)
mice was performed and the vaccines’ capacity to elicit high-
titer antigen specific systemic IgG antibodies against J8 or J14
was assessed using an enzyme-linked immunosorbent assay
(ELISA).
nostimulant lipid moiety (an adjuvant); and (3) multiple copies
of a B-cell epitope containing antigen. The carbohydrate cores
(4a, 4b; Scheme 1) were designed to be conjugated to the
immunostimulant lipid moiety at the anomeric position, with
the other carbohydrate hydroxyl groups conjugated to Boc-
protected amine containing spacers to enable the coupling of
multiple peptide epitopes to the carbohydrate carrier.
Carbohydrate core synthesis was initiated with the cyano-
ethylation of ꢀ-D-glucopyranosyl azide23 (1a) using acrylonitrile
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in acetonitrile
(Scheme 1). Acrylonitrile was freshly distilled before use to
prevent the formation of inseparable byproduct resulting from
its dimerization. Pure tetra-O-(cyanoethyl) D-glucopyranosyl
azide (2a; 66% yield), after selective reduction (catalytic
hydrogenation) of the azido group, was immediately conjugated
with freshly prepared monobenzyl adipate,24 using the peptide
coupling reagents, O-benzotriazole-N,N,N′,N′-tetramethyluro-
nium hexafluorophosphate (HBTU), and N,N-diisopropylethyl-
amine (DIPEA), to give benzyl ester 3a in 52% yield.
The ꢀ-D-galactopyranosyl derivatives (1b-4b; Scheme 1)
were prepared according to the procedure described by McGeary
et al.25 Problems were encountered, however, with the synthesis
of the ꢀ-D-glucopyranosyl core 4a. Reduction of the four
cyanoethyl groups to amines (using sodium borohydrate and
cobalt(II) chloride in methanol), t-butoxycarbonyl (Boc)-protec-
tion (with di-tert-butyl dicarbonate), and benzyl cleavage (by
saponification with lithium hydroxide) were performed in situ.
Due to the partial Boc-deprotection, caused by lowering the pH
of the aqueous phase during the separation of the product, the
formation of byproduct difficult to separate was observed. To
optimize the synthesis, catalytic hydrogenation over 10% Pd/C
instead of saponification with lithium hydroxide was done to
Results and Discussion
The described vaccine delivery system consists of three
components: (1) a carbohydrate core (a carrier); (2) an immu-

Liposaccharide-Based Vaccine DeliVery System
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1449
without adjuvant) and the positive control group (administered
J8 or J14 mixed with CFA; 6a and 6b vs J8+CFA, P > 0.05;
6c vs J14+CFA, P > 0.05), which demonstrates the potential
for using carbohydrate cores in the design of future subunit
vaccines. Moreover, the levels of specific serum IgG antibodies
elicited by vaccine candidates 6a or 6b were comparable to the
polylysine core-based vaccine 7 (6a and 6b vs 7, P > 0.05).
Changing the carbohydrate core did not have an effect on IgG
antibody levels (6a vs 6b, P > 0.05) as well as changing the
peptide antigen (6a vs 6c, P > 0.05; Figure 2).
Figure 1. Structure of polylysine core with J8 antigen (7).
reduce these problems. This resulted in higher yield of the ꢀ-D-
glucosyl derivative 4a, featuring a carboxylic acid at the
anomeric position (88% instead of 46%).
It has been previously demonstrated31,32 that both J8 and J14
peptides formulated with diphtheria toxoid and proteosomes,
respectively, are capable of inducing opsonic antibodies and
can protect outbred mice from virulent challenge. Therefore,
induction of opsonic antibodies, protective activity, and mucosal
immunization by liposaccharide vaccine candidates 6a-6c are
now under investigation. Success of this research could result
in a vaccine that not only prevents pharyngitis, but could save
many lives that are lost through RF and RHD, as it is believed
that they are associated with throat infection with GAS.
The lipid moiety used in the current study has been previously
demonstrated to enhance the elicitation of antigen-specific serum
IgG antibody responses against incorporated peptide anti-
gens2,13–15,26,27 The synthesis of the lipidic adjuvant was
performed on p-methylbenzhydrylamine (pMBHA) resin as
previously described.28 The lipid adjuvant consists of three
lipoamino acids (C12 ) 2-amino-D,L-dodecanoic acid) with
glycine spacers (C12-G-C12-C12-G). The Boc-protected glucosyl
(4a) and galactosyl (4b) carbohydrate cores were coupled to
the resin bound lipid adjuvant using HBTU/DIPEA in situ
neutralization.29 Couplings were repeated where necessary to
ensure a coupling efficiency greater than 99.6%, as confirmed
by quantitative ninhydrin analysis.30
Conclusion
The danger of rheumatic fever and the increasing number of
life-threatening invasive GAS infections in many countries of
the world lends support to attempts to develop safe and effective
vaccines against GAS infections. The development of self-
adjuvanting vaccines is emerging as a promising approach for
human vaccination in the future. Our vaccine delivery system
employs a glycolipid core acting as an adjuvant and a carrier
coupled to multiple copies of immunogenic peptides to induce
antibody responses without the coadministration of additional
potentially toxic adjuvants. Mice immunized with the described
liposaccharide vaccine candidates elicited comparable levels of
systemic J8-specific IgG antibodies to mice immunized with a
polylysine analogue. Although the fact that changing the
carbohydrate core did not have an effect on IgG antibody levels,
using different carbohydrate units as vaccine carriers provides
a unique possibility to engineer the vaccine system due to the
possibility to direct and change positions of peptide epitopes in
three-dimensional space and thus providing better peptide
recognition by cells and higher immune response. Moreover,
the results of this study suggest that this system could potentially
be used to develop vaccines against other infectious organisms,
and it could be potentially used to produce therapeutic vaccines,
which require cellular type immune responses.33
Synthetic peptide sequences, J8 or J14,22,31 were synthesized
by stepwise solid-phase peptide synthesis, and in situ neutraliza-
tion chemistry,29 following deprotection of the Boc-protected
carbohydrate amine groups. Finally, the products 6a, 6b, and
6c (Scheme 1) were cleaved from the resin using anhydrous
hydrofluoric acid, and purified by preparative reverse phase high
performance liquid chromatography (RP-HPLC) on a C4 column
using acetonitrile-water gradients containing 0.1% trifluoroacetic
acid over 60 min and characterized by liquid chromatography
electrospray ionization tandem mass spectrometry (LC-ESI-MS),
electrospray ionization mass spectrometry (ESI-MS), and ana-
lytical RP-HPLC.
As a positive control for the immunological studies a
convential polylysine lipid-core peptide system (7; Figure 1)
containing four copies of the J8 peptide antigen was produced.
Immunological assessment was performed in female B10.BR
(H-2k) mice. The mice (n ) 5/group) were injected subcutane-
ously on days 0, 21, 31, and 41 with 30 µg of compounds 6a,
6b, 6c, and 7 in phosphate-buffered saline (PBS). As positive
controls, two groups of mice were administered the peptide
antigens J8 or J14 emulsified in complete Freund’s adjuvant
(CFA). It has been previously reported that peptide antigens J8
and J14 did not elicit antibody response when administered
subcutaneously without CFA.15,31 For negative controls, mice
were administered CFA or PBS. Sera were collected from the
tail artery one day before each injection and nine days following
the last injection and then analyzed by ELISA for total antigen-
specific (J8 or J14) serum IgG antibodies. While all vaccine
candidates (6a, 6b, 6c, and 7) were unable to elicit significant
titers of J8 or J14-specific IgG antibodies following primary
immunization, a rapid increase in antigen-specific systemic IgG
antibody titers was observed following each boost (see Sup-
porting Information). At the final bleed (day 50), all mice
immunized with 6a-6c elicited high levels of specific serum
IgG antibodies (Figure 2). The vaccine structure–activity
response relationship was examined by comparing candidates
with different carbohydrate core (D-glucose or D-galactose) and
peptide epitope (J8 or J14) in reference to polylysine core (7),
see Figure 2. No significant difference was observed between
the levels of J8 or J14-specific serum IgG antibodies elicited
by carbohydrate core-based vaccines 6a-6c (administered
Experimental Section
Materials and Methods. All chemicals used in this study were
of analytical grade or equivalent unless otherwise stated. N-R-Boc-
L-amino acids and pMBHA resin were purchased from Novabio-
chem (Läufelfingen, Switzerland), Renanal (Budapest, Hungary)
and Peptides International (Louisville, Kentucky). Peptide synthesis
grade N,N′-dimethylformamide (DMF), dichloromethane (DCM),
trifluoroacetic acid (TFA), HBTU, and di-t-butyldicarbonate were
purchased from Auspep (Melbourne, VIC, Australia). HPLC grade
acetonitrile (ACN), isopropyl alcohol (IPA), and methanol (MeOH)
were supplied by Labscan (Dublin, Ireland) or Honeywell Burdick
and Jackson (Morristown, NJ). Hydrogen bromide in acetic acid
and ninhydrin were obtained from Merck (Kilsyth, VIC, Australia).
Palladium (10%) on carbon was purchased from Lancaster Synthesis
(Lancashire, England). Anhydrous hydrofluoric acid (HF) was
supplied by BOC gases (Sydney, NSW, Australia). 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. Flash chromatography was performed on silica

1450 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Simerska et al.
Figure 2. J8 or J14 specific serum IgG antibody titers (log10) at the final bleed (day 50) elicited in response to immunization of B10.BR (H-2k)
mice at the tail base with the carbohydrate core-based vaccine candidates (6a-6c) and the polylysine core-based vaccine (7), as determined by
ELISA. Statistical analysis was performed using a one-way ANOVA followed by the t-student posthoc test (no significant difference p > 0.05
between all vaccine candidates and their positive controls).
gel 60 (230–400 mesh; Lomb scientific, Taren Point, NSW,
Australia). Thin layer chromatography (TLC) was performed on
Merck (Darmstadt, Germany) aluminum-backed silica gel 60 F254
plates. Detection was achieved using 20% (v/v) H2SO4 in EtOH,
and compounds were revealed after heating. LC-ESI-MS and ESI-
MS were performed on a Perkin-Elmer-Sciex API3000 using the
Analyst 1.4 (Applied Biosystems/MDS Sciex, Toronto, Canada)
software. Samples (1–10 µL) were injected into ACN-H2O mobile
phases containing 0.1% (v/v) acetic acid and run in positive ion
the crude product was purified by flash chromatography on silica
(3:1 ethyl acetate–hexane) to give compound 2a as a white powder
(1.2 g; 2.9 mmol; 66% yield). Rf ) 0.53 (3:1 EtOAc-hexane).
ESI-MS: C18H23N7O5 (417.1839) m/z [M + H+]+ 418 (calcd,
+ +
418.2), [M + NH4
]
435.6 (calcd, 435.2), [M + Na+]+ 440.5
(calcd, 440.2). 1H NMR (300 MHz, CDCl3): δ 2.59–2.70 (m, 8H,
4 × CH2CN), 3.11 (t, 1H, H-3, J ) 8.8 Hz), 3.36 (t, 1H, H-2, J )
9.1 Hz), 3.42–3.48 (m, 1H, H-5), 3.55 (t, 1H, H-4, J ) 9.3 Hz),
3.70–4.18 (m, 10H, 5 × CH2O), 4.58 (d, 1H, H-1, J ) 8.6 Hz).
13C NMR (125 MHz, CDCl3): δ 19.0, 19.2, 19.3, 19.4, 66.3, 67.2,
67.5, 68.2, 76.4, 76.5, 76.6, 81.8, 83.9, 89.9, 118.0, 118.1, 118.5,
118.6.
1
mode. H NMR and 13C NMR spectra were recorded on either a
Bruker Avance 500 MHz spectrometer (Bruker Biospin, Germany)
or a Bruker Avance 300 MHz spectrometer (Bruker Biospin,
Germany) at 298 K in deuterated chloroform (CDCl3; Cambridge
Isotope Laboratories, Andover, MA). Chemical shifts are reported
in parts per million (ppm), with chemical shifts referenced to the
residual CHCl3 peak (δ 7.24 ppm). NMR spectra were processed
using Topspin 1.3 (Bruker Biospin, Germany). Analytical RP-HPLC
was performed using Shimadzu (Kyoto, Japan) instrumentation
(Class Vp 6.12 software, SCL-10AVp controller, SIL-10A auto-
injector, LC-10AT pump, LC-10AD pump, Waters 486 tunable
absorbance detector) using a 0–100% linear gradient of solvent B
over 30 min with a 1 mL/min flow rate and detection at 214 nm.
Solvent A consisted of 0.1% (v/v) aqueous TFA and solvent B
consisted of 90% ACN/H2O + 0.1% TFA (method 1) or 90% IPA/
H2O + 0.1% TFA (method 2). Separation was achieved on a Vydac
(Hesperia, CA) analytical C4 column (214TP54; 5 µm; 4.6 × 250
mm). Preparative RP-HPLC was performed on a Waters Delta 600
system in linear gradient mode using a 10 mL/minute flow rate,
with detection at 230 nm. Separations were performed on a Vydac
C4 preparative column (214TP1022; 10 µm; 22 × 250 mm).
6-(2,3,4,6-Tetra-O-(cyanoethyl)-ꢀ-D-glucopyranosylamino)-6-
oxohexanoic Acid Benzyl Ester (3a). Cyanoethylated ꢀ-D-glu-
copyranosyl azide 2a (4.2 g; 10 mmol) was dissolved in THF (260
mL) and hydrogenated at atmospheric pressure over 10% Pd/C (0.42
g) for 2 h. Upon completion (determined by TLC; eluent EtOAc),
adipic acid monobenzyl ester24 (2.6 g; 11 mmol), HBTU (3.99 g;
10.6 mmol), and DIPEA (2.1 mL; 12 mmol) were added to the
reaction mixture and stirred at room temperature under an argon
atmosphere for 16 h. The reaction mixture was then filtered through
Celite, which was thoroughly washed with methanol afterward. The
filtrate was condensed (rotary evaporation), dissolved in EtOAc (200
mL), and washed with 5% aqueous HCl (3 × 100 mL), saturated
aqueous NaHCO3 (2 × 100 mL), and saturated aqueous NaCl (50
mL), dried (MgSO4), and condensed (rotary evaporation). Pure
product 3a (3.17 g; 5.19 mmol; 52% yield) was obtained as colorless
oil after flash chromatography on silica (EtOAc). Rf ) 0.4 (EtOAc).
ESI-MS: C31H39N5O8 (609.2875) m/z [M + H+]+ 610.7 (calcd,
610.3), [M + NH4 ]
+ + 627 (calcd, 627.3), [M + Na+]+ 632 (calcd,
Synthesis of Carbohydrate Core 2,3,4,6-Tetra-O-(cyano-
ethyl)-ꢀ-D-glucopyranosyl Azide (2a). DBU (0.27 mL; 1.76
mmol) and freshly distilled acrylonitrile (2.88 mL; 43.7 mmol) were
added to a stirred solution of glucopyranosyl azide23 1a (0.9 g; 4.4
mmol) in acetonitrile (22.5 mL) at RT. After four hours, additional
portions of DBU (0.27 mL; 1.76 mmol) and acrylonitrile (2.88 mL;
43.7 mmol) were added, and the reaction was left to stir overnight.
The reaction mixture was then condensed (rotary evaporation), and
632.3). 1H NMR (300 MHz, CDCl3): δ 1.62–1.7 (m, 4H, CH2CH2),
2.25 (t, 2H, CH2CON, J ) 6.8 Hz), 2.36 (t, 2H, CH2COO, J ) 6.9
Hz), 2.50–2.66 (m, 8H, 4 × CH2CN), 3.23 (t, 1H, H6a, J ) 9.0
Hz), 3.36–3.42 (m, 2H, CH2O), 3.51 (t, 1H, H6b, J ) 9.5 Hz),
3.59–4.15 (m, 10H, 3 × CH2O, 4 × CHO), 5.03–5.06 (m, 1H,
H-1), 5.08 (s, 2H, CH2OPh), 6.47 (d, 1H, NH, J ) 9.5 Hz), 7.33
(br s, 5H, 5 × Ar-H). 13C NMR (125 MHz, CDCl3): δ 19.0, 19.2,
19.3, 19.4, 24.2, 24.5, 33.6, 36.1, 66.3, 67.4 (2 × ), 67.6, 68.0,

Liposaccharide-Based Vaccine DeliVery System
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1451
68.9, 74.0, 75.9, 76.9, 78.8, 81.5, 85.0, 118.4, 118.5, 118.6 (2×),
carbohydrate (4a or 4b) or poly lysine cores which were coupled
to the lipid core. Following Boc-deprotection, four copies of the
peptide antigen, J8 (QAEDKVKQSREAKKQVEKALKQLED-
KVQ) or J14 (KQAEDKVKASREAKKQVEKALEQLEDKVK),
were synthesized by stepwise solid phase peptide synthesis. After
the synthesis of each peptide was complete, the amino-terminal
Boc protecting groups were removed with TFA. The peptidyl-resins
were then thoroughly washed with DMF, DCM, and MeOH and
dried under vacuum prior to cleavage using anhydrous HF. HF
cleavage (10 mL HF/g resin) was performed at 0 °C for two hours
in the presence of 5% (v/v) p-cresol. Following the cleavage, the
HF was removed under reduced pressure, and the peptides were
precipitated in ice-cold diethyl ether, filtered, dissolved in 40%
ACN/H2O + 0.1% TFA, and lyophilized. The lyophilized products
were purified by preparative RP-HPLC on a C4 column using a
10–100% solvent B gradient over 60 min. The collected fractions
were analyzed by RP-HPLC and LC-ESI-MS and combined to give
6a (34 mg), 6b (24 mg), and 6c (38 mg) following lyophilization.
Compound 7 has been prepared and characterized as described
previously.36
Compound 6a: Purification yield, 57%; HPLC, tR ) 18.6 min
(method 1, C4 column), tR ) 17.2 min (method 2, C4 column);
LC-ESI-MS, [M + 13H+]13+ m/z 1101.2 (calcd, 1101.0), [M +
14H+]14+ m/z 1020.3 (calcd, 1022.4), [M + 15H+]15+ m/z 952.1
(calcd, 954.3), [M + 16H+]16+ m/z 892.7 (calcd, 895.0), [M +
17H+]17+ m/z 839.9 (calcd, 842.2); MW 14299.5 g/mol.
Compound 6b: Purification yield, 42%; HPLC, tR ) 20.0 min
(method 1, C4 column), tR ) 16.9 min (method 2, C4 column);
LC-ESI-MS, [M + 18H+]18+ m/z 795.1 (calcd, 795.4), [M +
19H+]19+ m/z 755.4 (calcd, 753.6), [M + 21H+]21+ m/z 684.4
(calcd, 682.0), [M + 23H+]23+ m/z 624.5 (calcd, 623.0), [M +
24H+]24+ m/z 596.5 (calcd, 596.0), [M + 26H+]26+ m/z 554.6
(calcd, 551.0); MW 14299.5 g/mol.
Compound 6c: Purification yield, 63%; HPLC, tR ) 18.7 (method
1, C4 column), tR ) 17.1 min (method 2, C4 column); LC-ESI-
MS, [M + 12H+]12+ m/z 1216.7 (calcd, 1214.1), [M + 15H+]15+
m/z 973.5 (calcd, 975.3), [M + 17H+]17+ m/z 862.2 (calcd, 859.0),
[M + 18H+]18+ m/z 809.9 (calcd, 811.0); MW 14587.92 g/mol.
Immunization Protocol. All protocols were approved by the
Queensland Institute of Medical Research Animal Ethics Committee
and were carried out according to Australian National Health and
Medical Research Council guidelines. Female B10.BR (H-2k) mice
(4–6 week-old, Animal Resource Centre, Perth, Western Australia,
Australia) were used for immunization. Mice (n ) 5/group) were
primed subcutaneously at the tail base with 30 µg of immunogens
(6a-6c) in a total volume of 50 µL of sterile-filtered PBS. Mice
received a further three boosts (30 µg of immunogens in a total
volume of 50 µL of PBS) at 10 day intervals (days 21, 31, and 41)
with. Two positive control groups received 30 µg of either J8 or
J14 emulsified in a total volume of 50 µL of CFA/PBS (1:1). Two
negative controls were administered a 50 µL of either CFA/PBS
(1:1) or PBS alone. All controls were boosted with 50 µL of PBS.
Blood was collected from the tail artery of each mouse one day
prior to each injection and nine days after the last immunization.
The blood was left to clot at 37 °C for 1 h and then centrifuged for
10 min at 3000 RPM to remove clots. Sera were then stored at
-20 °C.
Enzyme-Linked Immunosorbent Assay (ELISA). Determina-
tion of serum IgG antibodies against the J8 or J14 epitopes was
performed using a previously described ELISA.37 Optical density
was read at 450 nm in a microplate reader following the addition
of peroxidase-conjugated goat antimouse IgG, and O-phenylene-
diamine. The antibody titer was defined as the lowest dilution with
an optical density more than three standard deviations greater than
the mean absorbance of control wells containing normal mouse
serum.
Statistics. Statistical analysis of antibody titers was performed
using a one-way ANOVA followed by the t-student posthoc test.
GraphPad Prism 4 software was used for statistical analysis, with
p < 0.05 taken as statistically significant.
118.7, 128.1, 128.2, 128.6, 135.9, 173.4, 173.6.
6-(2,3,4,6-Tetra-O-(butoxycarbonylaminopropyl)-ꢀ-D-glucopy-
ranosylamino)-6-oxohexanoic Acid (5a). Compound 3a (1.0 g;
1.6 mmol), cobalt chloride hexahydrate (3.1 g; 13 mmol), and di-
tert-butyl dicarbonate (1.8 g; 8.3 mmol) were dissolved in methanol
(45 mL) and cooled to 0 °C (iced water). Aliquots of sodium
borohydride (2.48 g; 65.5 mmol) were then added to the mixture
over a 30 min period, and subsequently, the reaction was stirred
for 4 h at RT. Upon completion of the Boc-protection (determined
by TLC; eluent EtOAc), the mixture was filtered and evaporated
under reduced pressure to give crude compound 4a (1.5 g), which
was then dissolved in dry THF (80 mL), degassed, and hydroge-
nated over 10% Pd/C (120 mg) with stirring at room temperature
under H2(g) overnight. After evaporation, the crude product was
dissolved in 10% methanol in CHCl3, filtered through Celite,
concentrated, and purified by flash chromatography on silica using
the same eluent to give 5a (0.92 g; 0.99 mmol; 88% yield) as
colorless foam. Rf ) 0.3 (10% methanol in CHCl3). ESI-MS:
C44H81N5O16 (935.5757) m/z [M + H+]+ 936.9 (calcd, 936.6), [M
+ Na+]+ 959 (calcd, 958.6), [MH-BOC]+ 837 (calcd, 836.6).1H
NMR (300 MHz, CDCl3): δ 1.42 (br s, 36H, 4 × CMe3), 1.55–1.83
(m, 12H, 6 × CH2), 2.25–2.38 (m, 4H, 2 × CH2CO), 2.90–3.95
(m, 22H, 4 × CH2N, 5 × CH2O, 4 × CHO), 4.67 (br s, 1H, H-1),
4.77–5.22 (m, 5H, 5 × NH). 13C NMR (125 MHz, CDCl3): δ 24.3,
24.6, 24.9, 28.2, 28.4, 29.7, 29.9, 30.4, 30.6, 30.7, 33.4, 36.1, 38.2,
38.7, 41.0, 65.3, 66.0, 69.2, 70.2, 70.5, 70.8, 71.0, 71.4, 76.3, 77.2,
78.0, 79.0, 79.6, 79.8, 81.6, 85.2, 127.0, 127.6, 128.5, 156.0, 156.4,
156.6, 173.8.
Synthesis of Peptides and Lipopeptides. Lipopeptide vaccines
(6a-6c) were synthesized by stepwise solid-phase peptide synthesis
on pMBHA resin (0.4 mmol NH2/g; 0.2 mmol scale), using HBTU/
DIPEA in situ neutralization and Boc-chemistry.29 The pMBHA
resin was swollen in anhydrous DMF (10 mL/g of resin) for over
an hour followed by neutralization of the hydrochloride salt of the
resin-bound amine by 3 × 15 min treatments with 10% (v/v) DIPEA
in anhydrous DMF (10 mL/g of resin). Coupling cycles consisted
of Boc-deprotection (2 × 1 min treatments with neat TFA), a DMF
flow-wash (1 min), and couplings with 4.1 equiv of preactivated
amino acid (20–60 min). Amino acid activation (1 min) was
achieved by dissolving amino acids (0.82 mmol; 4.1 equiv) in a
0.5 M HBTU-DMF solution (1.6 mL; 0.8 mmol; 4 equiv), followed
by the addition of DIPEA (212 µL; 1.22 mmol). Coupling efficiency
was determined using the quantitative ninhydrin test,30 with
couplings repeated, if necessary, to give coupling yields greater
than 99.7%. Where coupling yields of greater than 99.7% could
not be achieved after three couplings, the remaining amines were
blocked by acetylation. This was achieved by treating the resin with
acetic anhydride (0.5 mL; 5.29 mmol), DIPEA (0.47 mL; 2.70
mmol), and DMF (14 mL) for 5 min, and then repeating the
treatment for 15 min. When coupling to a proline residue, the
chloranil test34 was used to qualitatively assess coupling yields to
the praline secondary amine. Boc-amino acids with the following
side-chain protection were utilized for the synthesis: Arg(Tos),
Asp(OcHx), Gln(Xan), Glu(OcHx), Lys(2-Cl-Z), Lys(Boc), Ser-
(Bzl). After the coupling of glutamine, the resin was washed with
DCM before and after Boc-deprotection to prevent high-tempera-
ture-catalyzed pyrrolidone carboxylic acid (Pca) formation.29
Synthetic racemic 2-(t-butoxycarbonylamino)-D,L-dodecanoic acid
(Boc-C12-OH)35 was utilized for lipid core synthesis. The lipid core
was synthesized by coupling Boc-Gly OH to pMBHA resin,
followed by two cycles of Boc-C12-OH, then Boc-Gly OH and Boc-
C12-OH. Carbohydrate cores (4a, 4b) were attached to the lipid
core on pMBHA resin (0.25 mmol NH2/g; 0.1 mmol scale) using
the same procedure as used for the coupling of amino acids. First,
activation of the carbohydrate cores 4a and 4b (1 min) was achieved
by dissolving 4a and 4b (0.2 mmol; 2 equiv) in a 0.5 M HBTU-
DMF (0.4 mL; 0.2 mmol; 2 equiv) followed by the addition of
DIPEA (70 µL; 0.4 mmol). Then, the activated carbohydrate cores
were added to the lipid core conjugated pMBHA resin and coupled
for 10 h. Four peptide attachment points were provided by the

1452 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Simerska et al.
(17) Carapetis, J. R.; Steer, A. C.; Mulholland, E. K.; Weber, M. The global
burden of group A streptococcal diseases. Lancet Infect. Dis. 2005, 5
(11), 685–694.
(18) Bisno, A. L.; Brito, M. O.; Collins, C. M. Molecular basis of group
A streptococcal virulence. Lancet Infect. Dis. 2003, 3 (4), 191–200.
(19) McArthur, J. D.; Walker, M. J. Domains of group A streptococcal M
protein that confer resistance to phagocytosis, opsonization and
protection: Implications for vaccine development. Mol. Microbiol.
2006, 59 (1), 1–4.
(20) Olive, C.; Batzloff, M. R.; Toth, I. Lipid core peptide technology and
group A streptococcal vaccine delivery. Exp. ReV. Vac. 2004, 3 (1),
43–58.
(21) Cunningham, M. W. Pathogenesis of group A streptococcal infections.
Clin. Microbiol. ReV. 2000, 13 (3), 470–511.
(22) Hayman, W. A.; Brandt, E. R.; Relf, W. A.; Cooper, J.; Saul, A.;
Good, M. F. Mapping the minimal murine T cell and B cell epitopes
within a peptide vaccine candidate from the conserved region of the
M protein of group A streptococcus. Int. Immunol. 1997, 9 (11), 1723–
1733.
(23) Gyorgydeak, Z.; Szilagyi, L.; Paulsen, H. Synthesis, Structure and
Reactions of Glycosyl Azides. J. Carbohydr. Chem. 1993, 12 (2), 139–
163.
(24) English, A. R.; Girard, D.; Jasys, V. J.; Martingano, R. J.; Kellogg,
M. S. Orally effective acid prodrugs of the beta-lactamase inhibitor
sulbactam. J. Med. Chem. 1990, 33 (1), 344–347.
(25) McGeary, R. P.; Jablonkai, I.; Toth, I. Carbohydrate-based templates
for synthetic vaccines and drug delivery. Tetrahedron 2001, 57 (41),
8733–8742.
Acknowledgment. This work was supported by the
Australian National Health and Medical Research Council
(NHMRC) and it is gratefully acknowledged. A.-B.M.A.-A.
would like to acknowledge financial support received through
the Egyptian Governments High Education Ministry PhD
funding scholarship. M.R.B. is supported by a postdoctoral
research fellowship from the National Heart Foundation of
Australia.
Supporting Information Available: Immunological data for
liposaccharide vaccine candidates 6a-6c and 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) O’Hagan, D. T.; Rappuoli, R. Novel approaches to pediatric vaccine
delivery. AdV. Drug DeliVery ReV. 2006, 58 (1), 29–51.
(2) Olive, C.; Toth, I.; Jackson, D. Technological advances in antigen
delivery and synthetic peptide vaccine developmental strategies. Mini-
ReV. Med. Chem. 2001, 1, 429–438.
(3) Mesa, C.; Fernandez, L. E. Challenges facing adjuvants for cancer
immunotherapy. Immunol. Cell Biol. 2004, 82 (6), 644–650.
(4) Bergeon, J. A.; Toth, I. Enhancement of oral drug absorptionsEffect
of lipid conjugation on the enzymatic stability and intestinal perme-
ability of l-Glu-l-Trp-NH2. Bioorg. Med. Chem. 2007, 15 (22), 7048–
7057.
(5) Falconer, R. A.; Toth, I. Design, synthesis and biological evaluation
of novel lipoamino acid-based glycolipids for oral drug delivery.
Bioorg. Med. Chem. 2007, 15 (22), 7012–7020.
(6) Pialoux, G.; Gahery-Segard, H.; Sermet, S.; Poncelet, H.; Fournier,
S.; Gerard, L.; Tartar, A.; Gras-Masse, H.; Levy, J. P.; Guillet, J. G.
Lipopeptides induce cell-mediated anti-HIV immune responses in
seronegative volunteers. AIDS 2001, 15 (10), 1239–1249.
(7) Livingston, B. D.; Crimi, C.; Grey, H.; Ishioka, G.; Chisari, F. V.;
Fikes, J.; Grey, H.; Chesnut, R. W.; Sette, A. The hepatitis B virus-
specific CTL responses induced in humans by lipopeptide vaccination
are comparable to those elicited by acute viral infection. J. Immunol.
1997, 159 (3), 1383–1392.
(26) Peter, M.; Moyle, C. O. M. F. G. I. T. Method for the synthesis of
highly pure vaccines using the lipid core peptide system. J. Pept. Sci.
2006, 12 (12), 800–807.
(27) Moyle, P. M.; Olive, C.; Karpati, L.; Barozzi, N.; Ho, M. F.; Dyer,
J.; Sun, H. K.; Good, M.; Toth, I. Synthesis and immunological
evaluation of M protein targeted tetra-valent and tri-valent group A
streptococcal vaccine candidates based on the lipid-core peptide
system. Int. J. Pept. Protein Res. 2006, 12 (3), 317–326.
(28) Horváth, A.; Olive, C.; Wong, A.; Clair, T.; Yarwood, P.; Good, M.;
Toth, I. A lipophilic adjuvant carrier system for antigenic peptides.
Lett. Pept. Sci. 2001, 8 (3), 285–288.
(29) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.
In situ neutralization in Boc-chemistry solid-phase peptide-
synthesissRapid, high-yield assembly of difficult sequences. Int. J.
Pept. Protein Res. 1992, 40 (3–4), 180–193.
(30) Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Quantitative
monitoring of solid-phase peptide synthesis by the ninhydrin reaction.
Anal. Biochem. 1981, 117 (1), 147–157.
(31) Batzloff, M. R.; Yan, H. R.; Davies, M. R.; Hartas, J.; Lowell, G. H.;
White, G.; Burt, D. S.; Leanderson, T.; Good, M. F. Toward the
development of an antidisease, transmission-blocking intranasal vac-
cine for group A streptococcus. J. Infect. Dis. 2005, 192 (8), 1450–
1455.
(8) Lechmann, M. P. D.; Jake Liang, T. M. D. Vaccine development for
hepatitis C. Semin. LiVer Dis. 2000, (02), 211–226.
(9) Benmohamed, L.; Thomas, A.; Bossus, M.; Brahimi, K.; Wubben, J.;
Gras-Masse, H.; Druilhe, P. High immunogenicity in chimpanzees of
peptides and lipopeptides derived from four new Plasmodium falci-
parum pre-erythrocytic molecules. Vaccine 2000, 18 (25), 2843–2855.
(10) Tam, J. P. Synthetic peptide vaccine design: Synthesis and properties
of a high-density multiple antigenic peptide system. Proc. Natl. Acad.
Sci. U.S.A 1988, 85 (15), 5409–5413.
(32) Batzloff, M. R.; Hayman, W. A.; Davies, M. R.; Zeng, M.; Pruksakorn,
S.; Brandt, E. R.; Good, M. F. Protection against group A streptococcus
by immunization with J8-diphtheria toxoid: Contribution of J8 and
diphtheria toxoid-specific antibodies to protection. J. Infect. Dis. 2003,
187 (10), 1598–1608.
(33) Moyle, P. M.; Olive, C.; Ho, M. F.; Pandey, M.; Dyer, J.; Suhrbier,
A.; Fujita, Y.; Toth, I. Toward the development of prophylactic and
therapeutic human papillomavirus type-16 lipopeptide vaccines.
J. Med. Chem. 2007, 50 (19), 4721–4727.
(34) Christensen, T. Qualitative test for monitoring coupling completeness
in solid-phase peptide-synthesis using chloranil. Acta Chem. Scand.
1979, 33 (10), 763–766.
(35) Gibbons, W. A.; Hughes, R. A.; Charalambous, M.; Christodoulou,
M.; Szeto, A.; Aulabaugh, A. E.; Mascagni, P.; Toth, I. Lipidic peptides
0.1. Synthesis, resolution and structural elucidation of lipidic amino-
acids and their homo-oligomers and heterooligomers. Liebigs Ann.
Chem. 1990, (12), 1175–1183.
(36) Olive, C.; Batzloff, M. R.; Horvath, A.; Wong, A.; Clair, T.; Yarwood,
P.; Toth, I.; Good, M. F. A lipid core peptide construct containing a
conserved region determinant of the group A streptococcal M protein
elicits heterologous opsonic antibodies. Infect. Immun. 2002, 70 (5),
2734–2738.
(37) Pruksakorn, S.; Galbraith, A.; Houghten, R. A.; Good, M. F. Conserved
T-cellandB-cellepitopesontheM-proteinofgroup-astreptococcisInduction
of bactericidal antibodies. J. Immunol. 1992, 149 (8), 2729–2735.
(11) Herrera, S.; de Plata, C.; Gonzalez, J. M.; Perlaza, B. L.; Bettens, F.;
Corradin, G.; Arevalo-Herrera, M. Antigenicity and immunogenicity
of multiple antigen peptides (MAP) containing P. vivax CS epitopes
in Aotus monkeys. Parasite Immunol. 1997, 19 (4), 161–170.
(12) Hayman, W. A.; Toth, I.; Flinn, N.; Scanlon, M.; Good, M. F.
Enhancing the immunogenicity and modulating the fine epitope
recognition of antisera to a helical group A streptococcal peptide
vaccine candidate from the M protein using lipid-core peptide
technology. Immunol. Cell Biol. 2002, 80 (2), 178–187.
(13) Moyle, P. M.; Olive, C.; Ho, M. F.; Good, M. F.; Toth, I. Synthesis
of a highly pure lipid core peptide based self-adjuvanting triepitopic
group A streptococcal vaccine, and subsequent immunological evalu-
ation. J. Med. Chem. 2006, 49 (21), 6364–6370.
(14) Horvath, A.; Olive, C.; Karpati, L.; Sun, H. K.; Good, M.; Toth, I.
Toward the development of a synthetic group A streptococcal vaccine
of high purity and broad protective coverage. J. Med. Chem. 2004,
47 (16), 4100–4104.
(15) Olive, C.; Hsien, K.; Horvath, A.; Clair, T.; Yarwood, P.; Toth, I.;
Good, M. F. Protection against group A streptococcal infection by
vaccination with self-adjuvanting lipid core M protein peptides.
Vaccine 2005, 23 (17–18), 2298–2303.
(16) Olive, C.; Hsien Kuo, S.; Mei-Fong, H.; Dyer, J.; Horvath, A.; Toth,
I.; Good, M. F. Intranasal administration is an effective mucosal
vaccine delivery route for self-adjuvanting lipid core peptides targeting
the group A streptococcal M protein. J. Infect. Dis. 2006, 194 (3),
316–324.
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