Chemical Synthesis of Proteins Using N-Sulfanylethylanilide Peptides, Based on N−S Acyl Transfer Chemistry

Abstract

Native chemical ligation (NCL), which features the use of peptide thioesters, is among the most reliable ligation protocols in chemical protein synthesis. Thioesters have conventionally been synthesized using tert-butyloxycarbonyl (Boc)-based solid-phase peptide synthesis (SPPS); however, the increasing use of 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS requires an efficient preparative protocol for thioesters which is fully compatible with Fmoc chemistry. We have addressed this issue by mimicking the naturally occurring thioester-forming step seen in intein-mediated protein splicing of the intein-extein system, using an appropriate chemical device to induce N-S acyl transfer reaction, avoiding the problems associated with Fmoc strategies. We have developed N-sulfanylethylanilide (SEAlide) peptides, which can be synthesized by standard Fmoc SPPS and converted to the corresponding thioesters through treatment under acidic conditions. Extensive examination of SEAlide peptides showed that the amide-type SEAlide peptides can be directly and efficiently involved in NCL via thioester species in the presence of phosphate salts, even under neutral conditions. The presence or absence of phosphate salts provided kinetically controllable chemoselectivity in NCL for SEAlide peptides. This allowed SEAlide peptides to be used in both one-pot/N-to-C-directed sequential NCL under kinetically controlled conditions, and the convergent coupling of large peptide fragments, which facilitated the chemical synthesis of proteins over about 100 residues. The use of SEAlide peptides, enabling sequential NCL operated under kinetically controlled conditions, and the convergent coupling, were used for the total chemical synthesis of a 162-residue monoglycosylated GM2-activator protein (GM2AP) analog.