Chemical synthesis of proteins with non-strategically placed cysteines using selenazolidine and selective deselenization

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125-residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C-terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N-terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analogue in which the active-site histidine was substituted with the unnatural and isosteric amino acid β-thienyl-l-alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site. Stitching a protein together: A synthesis approach is reported using selenazolidine and deselenization to access a protein with non-strategically placed cysteine residues. The challenging human phosphohistidine phosphatase 1 (PHPT1) protein, a 125-residue enzyme with three cysteine residues near the C-terminus, was used as a model system.