Native Chemical Ligation at Phenylalanine via Ortho-Mercaptophenylalanine

Native chemical ligation (NCL) enables the synthesis of complex peptides and proteins. Prior work in NCL at phenylalanine (Phe) employed β-mercapto or β-seleno (alkyl) derivatives. Herein, we describe an alternative approach to NCL at Phe, via an aryl thiol on the novel Phe surrogate ortho-mercaptophenylalanine (2-mercaptophenylalanine). Commercially available Boc-2-iodo-phenylalanine was incorporated at the N-terminus of the peptide. 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 (pK_a = 5.1, substantially lower than alkyl thiols). Alternatively, 2-mercaptophenylalanine could be incorporated via the short synthesis of Boc-2-(S-tert-butyl)mercaptophenylalanine and coupling to peptide on resin. Peptides containing 2-mercaptophenylalanine rapidly underwent NCL reactions with peptides containing C-terminal thioesters. NCL reactions with the thioesters of Gly, Ala, Leu, and Phe were complete in 5 min1 h at room temperature at 0.52 mM peptide concentrations. Reactions with 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. 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 NCL at phenylalanine.