CHEMICAL SYNTHESIS OF PROTEINS WITH NON-STRATEGICALLY PLACED CYSTEINES USING SELENAZOLIDINE AND SELECTIVE DESELENIZATION

Chemical synthesis of proteins has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. One particularly challenging protein is a 125-residue human phosphohistidine phosphatase 1 (PHPT1), whose three cysteines near the protein’s C-terminus are not strategically placed for ligation. Here, 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 ligations, deselenization provided the native alanine residue. This approach was used to synthesize both the wild-type PHPT1 and an analog 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’s active site.