Native Chemical Ligation at Phenylalanine via 2-Mercaptophenylalanine

Native chemical ligation (NCL) enables the synthesis of complex peptides and proteins with defined post-translational modifications and/or non-encodable incorporated functionalities. Herein, we describe a practical approach to native chemical ligation at phenylalanine (Phe), via the Phe surrogate 2-mercaptophenylalanine. The commercially available amino acid Boc-2-iodo-phenylalanine was used as a starting material and incorporated at the N-terminus of the peptide via standard solid-phase peptide synthesis. A solid-phase copper-mediated cross-coupling reaction with thioacetic acid was conducted on fully synthesized peptides with an N-terminal Boc-2-I-Phe to introduce, after TFA cleavage and deprotection, a nucleophilic aryl thiolate in the resultant N-terminal residue 2-mercaptophenylalanine (pKa = 5.1). Alternatively, 2-mercaptophenylalanine could be incorporated at the N-terminus via the short synthesis of Boc-2-(S-tert-butyl)-mercaptophenylalanine and coupling to peptide on resin. Peptides containing this amino acid rapidly underwent native chemical ligation reactions with peptides containing C-terminal thioesters. NCL reactions with the thioesters of Gly, Ala, Leu, and Phe were complete in 5 min-1 hour at room temperature at 0.5-2 mM peptide concentrations. Reactions with the more sterically demanding Val and Pro thioesters also proceeded in high yield, in 6 h and 12 h, respectively. NCL reactions with 2-mercaptophenylalanine also proceeded efficiently at pH 5. The peptide ligation product was desulfurized using nickel boride to generate phenylalanine at the ligation site. These results, using commercially available reagents and the possibility of amino acid synthesis on the solid phase which eliminates the requirement for solution-phase amino acid synthesis, represent a practical approach to native chemical ligation at phenylalanine.