Fmoc-Based Synthesis of Peptide-αThioesters: Application to the Total Chemical Synthesis of a Glycoprotein by Native Chemical Ligation

The technique of native chemical ligation has enabled the total chemical synthesis of proteins with molecular weights far in excess of those achievable by conventional stepwise solid-phase peptide synthesis. The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal α thioester and the other an N-terminal cysteine residue, to afford 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 synthesis of a glycosylated protein, the antimicrobial O -linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-residue glycopeptide- α thioester segment, which was complicated by the incompatibility of glycosidic linkages with Boc chemistry and by the incompatibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide “safety-catch” linker circumvented this problem and permitted the solid-phase synthesis of the glycopeptide- α thioester using standard Fmoc chemistry protocols. Ligation of this thioester with a 58-residue glycopeptide bearing 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.