An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.

A selective bi-directional peptide bond cleavage mediated by N-methylcysteine (MeCys) in Xaa-MeCys-Yaa peptides (Xaa and Yaa, non-cysteine residues) leading to thioesters and thiolactones is described. Rate and product analyses showed that an N^α-amide bond cleavage occurred at the Xaa-MeCys bond by an N–S acyl shift to generate an Xaa-S-(MeCys-Yaa) thioester at pH 1–5, whereas under strongly acidic conditions of H₀ = –5, the MeCys-Yaa bond underwent a C^α-amide bond cleavage via an oxazolone intermediate, which was trapped by thiocresol (TC) as an Xaa-MeCys-TC thioester. This thioester was then transformed into an Xaa-MeCys-β-thiolactone at pH 4–5. Replacing MeCys by a Cys residue did not result in significant bi-directional peptide bond cleavage, which suggests that N-methylation in a MeCys residue is important for the N–S acyl shift reaction and formation of oxazolone. The isomerization of amides and thioesters was successfully used to prepare cyclic peptides.