Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope

Article abstract of DOI:10.1016/j.bmcl.2008.12.013

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilisin

Full text of DOI:10.1016/j.bmcl.2008.12.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 821–824
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a Streptococcus pyogenes vaccine candidate based
on the M protein PL1 epitope
*
Pavla Simerska , Hao Lu, Istvan Toth
The University of Queensland, School of Molecular and Microbial Sciences (SMMS), Chemistry Building 68, St. Lucia, Brisbane, Qld 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the
bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino
acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis
procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray
ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate
may contribute to the discovery of a therapeutically and clinically viable vaccine against group A
streptococcus.
Received 7 November 2008
Revised 28 November 2008
Accepted 3 December 2008
Available online 7 December 2008
Keywords:
Streptococcus pyogenes
Self-adjuvanting vaccine
PL1 peptide epitope
Carbohydrate carrier
Ó 2008 Elsevier Ltd. All rights reserved.
Group A streptococcus (GAS, Streptococcus pyogenes) is respon-
sible for a range of infectious conditions, ranging from mild tonsil-
litis to more severe form of necrotizing fasciitis. Despite successful
treatment of these infections with adequate doses of antibiotics,
sequential post-infectious autoimmune responses, including rheu-
matic heart disease (RHD) and rheumatic fever (RF), still affect mil-
lions of lives each year.1 To avoid pandemic situations and to
reduce the fatality rate of both GAS infections and post-streptococ-
cal diseases, initial preventative measures to impede primary
infections caused by GAS bacteria need to be considered. Vaccina-
tion, remains one of the most effective and cost-efficient methods
in infectious disease prevention and control, and thus is the pre-
ferred approach to managing GAS infections.1,2
Antigenic epitopes attached to a peptide-based carrier and
a lipid moiety were shown by previous studies to be highly immu-
nogenic and generate comparable titres of antibodies against
incorporated epitopes when compared to the same epitopes
administered with an adjuvant (e.g., complete Freund’s adjuvant,
CFA).7–10 The presence of lipoamino acids (LAAs) resulted in an in-
creased lipophilic character of the construct which contributed to
enhancing the uptake of the vaccine candidate and also acted as
a built in adjuvant. The co-administration of alternative and often
toxic adjuvants (e.g., CFA or cholera toxins) was not necessary. This
novel adjuvant system also opens the way towards alternative vac-
cination routes such as the intranasal or oral routes.1,8,10
The synthesis of the carbohydrate carrier (Scheme 1) involved
The aim of this project was to synthesise a peptide subunit vac-
cine candidate against GAS; utilising peptide, carbohydrate and
lipid moieties. Four copies of an antigenic peptide epitope known
as PL1 were incorporated into the carbohydrate core of the vaccine
construct. PL1 is a peptide epitope derived from the variable N-ter-
minus region of the antigenic M-protein of a virulent GAS serovar,
the cyanoethylation11,12 of b-
D
-glucopyranosyl azide (1) giving tet-
ra-O-(cyanoethyl)-D-glucopyranosyl azide (2) followed by selective
hydrogenation of the azido group of compound 2 in dry tetrahy-
drofuran (THF) in the presence of palladium. The resulting amino
group was then coupled to monobenzyl adipate using O-benzotri-
azole-N,N,N0,N0-tetramethyluronium hexafluorophosphate (HBTU)
and N,N-diisopropylethylamine (DIPEA) in THF.13 The four cyano-
ethyl groups of the benzyl ester derivative 4 were immediately
reduced to primary amines via the addition of sodium borohydride
(NaBH4) and a catalytic amount of cobalt chloride (CoCl2) and then
Boc-protected to yield ester 5. The benzyl group of compound 5
was cleaved by reductive hydrogenation (H2 and Pd/C), resulting
this a
-helical surface protein present in most GAS strains.1,3,4 PL1
epitope was shown1,3 to instigate high levels of immune response
against specified serotypes of the GAS family and it was reported4–6
that multiple copies of peptide epitope elicited higher antibody
titres than a single peptide unit. The carbohydrate core acts as a
carrier of peptide epitopes and the lipoamino acid-based unit acts
as an adjuvant (Fig. 1).
in a carboxylic acid at the anomeric position of b-D-glucosyl deriv-
ative 6. Yields were found to be much higher when performing the
reaction with a hydrogenator (Thales H-Cube Hydrogenator, Thales
Nanotechnology, Hungary) rather than under standard procedure
(H2 atmosphere).14
* Corresponding author. Tel.: +61 7 33469892; fax: +61 7 33654273.
E-mail address: p.simerska@uq.edu.au (P. Simerska).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.013

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P. Simerska et al. / Bioorg. Med. Chem. Lett. 19 (2009) 821–824
Figure 1. Structure of the vaccine candidate against group A streptococcus.
Scheme 1. Synthesis of the
D-glucose-based carrier. Reagents and conditions: (a) MeCN, acrylonitrile, DBU (b) THF, H2, 10% Pd/C, rt; (c) HBTU, DIPEA, THF, monobenzyl
adipate, rt; (d) NaBH4, CoCl2, Boc2O, rt; (e) hydrogenation (Thales H-Cube Hydrogenator).
Scheme 2. Synthesis of the vaccine construct-utilising SPPS procedures. Reagents and conditions: (f) coupling of Gly, C12, C12 Gly and C12 and carbohydrate core 6 onto
pMBHA resin in the presence of HBTU (1.5 equiv) and DIPEA (1.6 equiv) in DMF, Boc-protecting groups were removed with TFA; (g) coupling of four copies of the PL1 using
HBTU, DIPEA in DMF; Boc-deprotection by TFA; cleavage of crude product from the pMBHA resin using HF and p-cresol scavenger.

P. Simerska et al. / Bioorg. Med. Chem. Lett. 19 (2009) 821–824
823
Figure 2. Characterisation of the pure GAS vaccine candidate (9) by analytical RP-HPLC and ESI-MS results. Reagents and conditions: (I) RP-HPLC eluent A—0.1% TFA/H2O;
eluent B—90% MeCN/10% H2O/0.1% TFA; 0–100% gradient of the eluent B over 30 min, 1 mL/min flow rate, 214 nm; Vydac C4 column (214TP54, 5
m, 4.6 ꢀ 250 mm); (II) RP-
HPLC eluent A—0.1% TFA/H2O; eluent B—90% MeOH/10% H2O/0.1% TFA; 0–100% gradient of the eluent B over 30 min, 1 mL/min flow rate, 214 nm; Vydac C4 column
(214TP54, 5
m, 4.6 ꢀ 250 mm); (III) ESI-MS—multiple charged ions from the vaccine molecule (Mw = 10,316.8 g/mol).
l
l
3. Brandt, E. R.; Hayman, W. A.; Currie, B.; Pruksakorn, S.; Good, M. F. Vaccine
1997, 15, 1805.
4. Brandt, E. R.; Teh, T.; Relf, W. A.; Hobb, R. I.; Good, M. F. Infect. Immun. 2000, 68,
6587.
5. Brandt, E. R.; Good, M. F. Immunol. Res. 1999, 19, 9.
6. Brandt, E. R.; Sriprakash, K. S.; Hobb, R. I.; Hayman, W. A.; Zeng, W.; Batzloff, M.
R.; Jackson, D. C.; Good, M. F. Nat. Med. 2000, 6, 455.
7. McGeary, R. P.; Jablonkai, I.; Toth, I. Tetrahedron 2001, 57, 8733.
8. McGeary, R. P.; Olive, C.; Toth, I. J. Pept. Sci. 2003, 9, 405.
9. Horvath, A.; Olive, C.; Karpati, L.; Sun, H. K.; Good, M.; Toth, I. J. Med. Chem.
2004, 47, 4100.
The lipid moiety 7 was generated using stepwise solid-phase
peptide synthesis (SPPS) (Scheme 2), where three C12 LAAs and
two glycine spacers were coupled to p-methylbenzhydrylamine
(pMBHA) resin. The same methods were applied for further cou-
pling of the D-glucose carrier 6 to lipid moiety 7 to form liposaccha-
ride 8. Upon removal of the Boc-protecting groups, four copies of
PL1 epitope (EVLTR RQSQD PKYVT QRIS)15 were coupled onto the
liposaccharide 8. Coupling efficiencies during the SPPS procedures
were checked after coupling steps by ninhydrin test, ensuring that
a minimal 99.6% coupling efficiency was achieved (if necessary
double coupling was employed).16,17 Upon completion of the syn-
thesis (Scheme 2), the crude vaccine candidate 9 was cleaved from
the pMBHA resin using anhydrous HF.18
The crude product 9 was purified by preparative reversed-phase
HPLC (RP-HPLC) and its purity was confirmed by analytical RP-
HPLC and electrospray ionisation mass spectrometry (ESI-MS).
The retention times of the pure compound 9 were detected to be
18.1 and 24.2 min using conditions A (Fig. 2, I) and B (Fig. 2, II),
respectively, and the structure of the purified product was con-
firmed by ESI-MS (Fig. 2, III).19
10. Horvath, A.; Olive, C.; Wong, A.; Clair, T.; Yarwood, P.; Good, M.; Toth, I. J. Med.
Chem. 2002, 45, 1387.
11. Materials and reagents: All materials and chemicals used in the experiments
were of analytical grade or equivalent. Boc-protected-L-amino acids and
pMBHA resin were purchased from Novabiochem (Laufelfingen, Switzerland),
Renanal (Budapest, Hungary), and Peptides International (Louisville,
Kentucky). Peptide synthesis reagents such as N,N0-dimethylformamide
(DMF), dichloromethane (DCM), HBTU, TFA, and di-tert-butyldicarbonate
were purchased from Auspep (Melbourne, Vic., Australia). Acetonitrile (ACN),
isopropyl alcohol (IPA), and MeOH, used during RP-HPLC, were purchased from
Labscan (Dublin, Ireland). Hydrogen bromide utilised during the ninhydrins
assay were supplied by Merck (Kilsyth, Vic., Australia). Hydrofluoric acid (HF),
used for peptide–resin cleavage, was purchased from BOC gases (Sydney, NSW,
Australia). All other chemicals were supplied by Sigma–Aldrich (Castle Hill,
NSW, Australia).
12. Simerska, P.; Abdel-Aal, A. M.; Fujita, Y.; Moyle, P. M.; McGeary, R. P.; Batzloff,
M. R.; Olive, C.; Good, M. F.; Toth, I. J. Med. Chem. 2008, 51, 1447.
The physiological efficacy of the synthesised compound 9 will
be evaluated in vivo in mice and reported elsewhere.
13. 6-(2,3,4,6-Tetra-O-(cyanoethyl)-b-
D-glucopyranosylamino)-6-oxohexanoic
acid
benzyl ester (3): Cyanoethylated azide (2) (1.40 g, 3.33 mmol) was
hydrogenated under atmospheric pressure over 10% Pd/C (0.14 g) in THF
(88 mL) for 2 h. Thin layer chromatography (TLC, aluminium-backed silica gel
60 F254 plates) was performed to determine the degree of completion of the
reaction using ethyl acetate (EtOAc) as eluent. For detection, mixture of 20% (v/
v) H2SO4 in EtOH was used. Adipic acid monobenzyl ester (0.83 g; 3.51 mmol),
HBTU (1.34 g, 3.56 mmol), and DIPEA (0.62 g, 4.76 mmol) were added to the
reaction mixture and stirred at room temperature under argon atmosphere for
24 h. The crude mixture was filtered through Celite and washed with MeOH.
After condensation (rotary evaporation), the filtrate was dissolved in EtOAc
(200 mL) and washed with HCl (5% aq, 3ꢀ 100 mL), NaHCO3 (aq, 2ꢀ 100 mL),
and saturated NaCl (aq, 50 mL). The mixture was dried over MgSO4
and condensed. The product 3 was obtained (1.51 g, 2.54 mmol) in 76.13%
yield.
Acknowledgements
This work was financially supported by the Australian National
Health and Medical Research Council (NHMRC). The author
acknowledges the encouragements and supports provided by all
parties.
References and notes
1. Olive, C.; Batzloff, M. R.; Toth, I. Expert Rev. Vaccine 2004, 3, 43.
2. Olive, C.; Toth, I.; Jackson, D. Mini-Rev. Med. Chem 2001, 1, 429.

824
P. Simerska et al. / Bioorg. Med. Chem. Lett. 19 (2009) 821–824
D-glucopyranosylamino)-6-
ninhydrin test to ensure 99.6% coupling efficiency. SPPS procedures were
14. 6-(2,3,4,6-Tetra-O-(butoxycarbonylaminopropyl)-b-
oxohexanoic acid (5): Compound
3
(1.51 g, 2.54 mmol), cobalt chloride
repeated until coupling efficiency was achieved. Lipid moiety was
synthesised in the sequence of Boc-Gly-OH, 2ꢀ Boc-C12-OH, Boc-Gly-OH
and Boc-C12-OH. The activated carbohydrate core 6 was then coupled to
lipid moiety 7. Following successful coupling, four copies of PL1 (EVLTR
RQSQD PKYVT QRIS) peptide epitope, were coupled by stepwise SPPS to the
liposaccharide 8.
hexahydrate (2.84 g; 11.90 mmol), and di-tert-butyl dicarbonate (1.63 g,
7.98 mmol) were dissolved in MeOH (40 mL, 0 °C). Sodium borohydride
(2.26 g, 59.70 mmol) was added to the reaction mixture and stirred for 2 h at
rt. TLC (eluent EtOAc) was used to determine the degree of completion and the
crude mixture was filtered to elude crude compound 4, which was then
dissolved in THF, the benzyl group was cleaved via hydrogenation. Upon
17. Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 177,
147.
18. Hydrofluoric acid (HF) cleavage procedures: Completed vaccine construct was
cleaved from pMBHA resin using anhydrous HF (Kel-F HF apparatus, Peptide
Institute, Osaka, Japan). Caution: Highly hazardous to health. The procedure
was performed at 0 °C for 3 h. p-Cresol (5%, v/v) was also present during the
completion (checked with TCL, eluent EtOAc), crude product
1.18 mmol, 46.40% yield) was condensed via rotary evaporation.
15. Moyle, P. M.; Olive, C.; Good, M.; Toth, I. J. Pept. Sci. 2006, 12, 800.
16. Solid-phase peptide synthesis (SPPS): The vaccine construct
5 (1.10 g,
9
was
synthesised using SPPS procedures and Boc-chemistry. pMBHA resin
(0.40 mmol, 0.12 mmol scale), was swelled in DMF for 2 h followed by
in situ neutralisation with DIPEA (10% v/v, 3ꢀ 15 min) in DMF. SPPS
cleaving process as
a scavenger. Upon completion, the product 9 was
precipitated (diethyl ether), filtered, dissolved (40% ACN/H2O) and
procedures involved
a
cycle of Boc-deprotection with TFA (2ꢀ 1-min
lyophilised to elicit the crude vaccine candidate (9).
treatments), flow-wash with DMF, and addition of pre-activated amino
acids (4.4 equiv, 60–100 min). Amino acids were activated by mixing with
0.5 M HBTU–DMF solution (2.56 mL, 1.28 mmol, 4 equiv) and DIPEA
(0.33 mL; 1.92 mmol). The coupling efficiency was checked with
19. Vaccine Construct (9): LC–ESI-MS, [M+9H+]9+ m/z 1155.4 (calcd, 1147.3),
[M+10H+]10+ m/z 1040.1 (calcd, 1032.7), [M+11H+]11+ m/z 945.8 (calcd,
938.9), [M+12+]12+ m/z 860.7 (calcd, 866.8), [M+13H+]13+ m/z 800 (calcd,
794.6), [M+14H+]14+ m/z 743.2 (calcd, 737.9); Mw 10,316.8 g/mol.