Copper-Mediated Selenazolidine Deprotection Enables One-Pot Chemical Synthesis of Challenging Proteins

While chemical protein synthesis has granted access to challenging proteins, the synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine residues, which are essential for native chemical ligations, as well as multiple purification and isolation steps. We describe the one-pot total synthesis of human thiosulfate:glutathione sulfurtransferase (TSTD1). WT-TSTD1 was synthesized in a C-to-N synthetic approach involving multiple NCL reactions, Cu^II -mediated deprotection of selenazolidine (Sez), and chemoselective deselenization. The seleno-analog Se-TSTD1, in which the active site Cys is replaced with selenocysteine, was also synthesized with a kinetically controlled ligation with an N-to-C synthetic approach. The catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1, thus suggesting that selenoproteins can have physiologically comparable sulfutransferase activity to their cysteine counterparts.