Fmoc-based synthesis of peptide-(alpha)thioesters: Application to the total chemical synthesis of a glycoprotein by native chemical ligation

The technique of native chemical ligation has enabled the total chemical sy nthesis of proteins with molecular weights far in excess of those achievabl e by conventional stepwise solid-phase peptide synthesis. The method involv es the condensation of two unprotected peptide segments, one bearing a C-te rminal (alpha)thioester and the other an N-terminal cysteine residue, to af ford a protein with a native amide linkage at the site of ligation. Here we report an extension of the native chemical ligation method to the total sy nthesis of a glycosylated protein, the antimicrobial O-linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-re sidue glycopeptide-(alpha)thioester segment, which was complicated by the i ncompatibility of glycosidic Linkages with Boc chemistry and by the incompa tibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide " safety-catch" linker circumvented this problem and permitted the solid-p hase synthesis of the glycopeptide-athioester using standard Fmoc chemistry protocols. Ligation of this thioester with a 58-residue glycopeptide beari ng an N-terminal cysteine residue yielded the full-length glycoprotein with two sites of glycosylation. The fully deprotected diptericin glycoform was active in antimicrobial assays.