An efficient peptide ligase with exquisite site-specificity is highly desirable for modifying peptides and proteins. Recently, we discovered a novel Asn/Asp-specific ligase called butelase 1 from the medicinal plant Clitoria ternatea. It is the fastest known ligase with high catalytic efficiency for catalyzing intramolecular cyclizations. However, lntermolecular ligations by butelase 1 would require an excess amount of substrate, enzyme or both to compete with the leaving groups to afford the ligation reaction with high efficiency, a feature shared by other ligases for intermolecular ligations. Here we describe the use of thiodepsipeptide substrates with thiols as leaving groups and unacceptable nucleophiles to render the butelase-mediated ligation reactions irreversible and in high yield. Butelase 1 also accepted desipeptide as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol as a poor nucleophile. The thiodesipeptide method for butelase-mediated intramolecular ligation was successfully applied for modilfying ubiquitin and green fluorescent protein with exquisite site-specificity and in high yields.