Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein

Starting from the α-(2,4-diniethoxybcnzyl) ester of N-(9-fluorenylniethoxycarbonyl)aspartic acid [Fmoc-Asp-ODmb], side-chain-protected resin-bound Fmoc-peptides containing an N^c-l-(4,4-dimethyl-2,6-dioxocyclohexylidenc)ethyl lysY1 [Lys(Dde)] residue were prepared. The C-terminal dimethoxybenzyl esters of aspartic acid were removed with 1% trifluoroacetic acid and 10% anisole in dichloromethane, followed by Fmoc-cleavage in the usual manner. The resin-bound peptides were then cyclized using 1-benzotriazolyloxy-tris-[N-pyrrolidino]phosphonium hexafluorophosphate (PyBOP) in the presence of N-methylmorpholine. The (dimethyldioxocyclohexylidene)ethyl groups of lysine were removed with 1% hydrazine hydrate in N,N-dimethylacetarnide, and the liberated side-chain amino functions were modified by reaction with pentafluorophenyl S-acetylinercaptoacetate (SAMA-OPfp). Finally, the peptides were side-chain deprotected, with exception of the Lys(SAMA) residue. and cleaved from the solid support with trifluoroacetic acid/anisole/ water, 95/2.5/2.5. Cyclic peptides comprising 7–14 amino acid residues were obtained employing this procedure. As a model conjugation. cyclo[Thr-Asn-Asn-Asn-Leu-Lys(SAMA)-Thr-Lys-Asp] was coupled with bromoacetamide. The same peptide was also coupled with a bromoacetylpeptide to give a well defined peptide1 peptide conjugate. All peptides were conjugated to bromoacetylated tetanus toxoid for immunization purposes.