Copper-mediated selenazolidine deprotection enables one-pot chemical synthesis of challenging proteins

While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts.