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

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 3, 2009, Pages 821-824

  Simerska, Pavla ^a   Lu, Hao ^a   Toth, Istvan ^a  

^a UNIVERSITY OF QUEENSLAND   (Australia)

Author keywords

Carbohydrate carrier; PL1 peptide epitope; Self adjuvanting vaccine; Streptococcus pyogenes

Indexed keywords

ADJUVANT; AMINO ACID; BACTERIAL VACCINE; CARBOHYDRATE; EPITOPE; M PROTEIN; PROTEIN PL1; STREPTOCOCCUS PYOGENES VACCINE; UNCLASSIFIED DRUG;

ARTICLE; DRUG SYNTHESIS; ELECTROSPRAY MASS SPECTROMETRY; NONHUMAN; PEPTIDE SYNTHESIS; REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; SOLID PHASE SYNTHESIS; STREPTOCOCCUS GROUP A;

AMINO ACIDS; ANTIGENS; ANTIGENS, BACTERIAL; BACTERIAL PROTEINS; BACTERIAL VACCINES; CARBOHYDRATES; CHROMATOGRAPHY, HIGH PRESSURE LIQUID; EPITOPES; HUMANS; LIPIDS; MODELS, CHEMICAL; SPECTROMETRY, MASS, ELECTROSPRAY IONIZATION; STREPTOCOCCAL VACCINES; STREPTOCOCCUS; STREPTOCOCCUS PYOGENES;

POSIBACTERIA; STREPTOCOCCUS PYOGENES; STREPTOCOCCUS SP. 'GROUP A';

EID: 58549118912     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.12.013     Document Type: Article

References (19)

1. Olive C., Batzloff M.R., and Toth I. Expert Rev. Vaccine 3 (2004) 43
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8. McGeary R.P., Olive C., and Toth I. J. Pept. Sci. 9 (2003) 405
9. Horvath A., Olive C., Karpati L., Sun H.K., Good M., and Toth I. J. Med. Chem. 47 (2004) 4100
10. Horvath A., Olive C., Wong A., Clair T., Yarwood P., Good M., and Toth I. J. Med. Chem. 45 (2002) 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,N′-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., and Toth I. J. Med. Chem. 51 (2008) 1447
13. 4 and condensed. The product 3 was obtained (1.51 g, 2.54 mmol) in 76.13% yield.
14. 6-(2,3,4,6-Tetra-O-(butoxycarbonylaminopropyl)-β-d-glucopyranosylamino)-6-oxohexanoic acid (5): Compound 3 (1.51 g, 2.54 mmol), cobalt chloride 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 completion (checked with TCL, eluent EtOAc), crude product 5 (1.10 g, 1.18 mmol, 46.40% yield) was condensed via rotary evaporation.
15. Moyle P.M., Olive C., Good M., and Toth I. J. Pept. Sci. 12 (2006) 800
16. Solid-phase peptide synthesis (SPPS): The vaccine construct 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, 3× 15 min) in DMF. SPPS procedures involved a cycle of Boc-deprotection with TFA (2× 1-min 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 ninhydrin test to ensure 99.6% coupling efficiency. SPPS procedures were repeated until coupling efficiency was achieved. Lipid moiety was synthesised in the sequence of Boc-Gly-OH, 2× 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.
17. Sarin V.K., Kent S.B.H., Tam J.P., and Merrifield R.B. Anal. Biochem. 177 (1981) 147
18. 2O) and lyophilised to elicit the crude vaccine candidate (9).
19. 14+ m/z 743.2 (calcd, 737.9); Mw 10,316.8 g/mol.