The procedure for converting peptide hydrazides into the corresponding peptide alkyl thioesters, based on this concept, is outlined in detail in Fig. . Initially, peptide acyl azide 2 was generated from peptide hydrazide 1 at pH 3 and -15°C. Subsequently, the thiolysis step, leading to peptide aryl thioester 4, was initiated by adding an excess of aryl thiol 3 and adjusting the pH to 7. After 15 minutes at room temperature (rt), the thiol-thioester exchange reaction leading to the formation of peptide alkyl thioester 6 was triggered by adding alkyl thiol 5 (2 equiv) and TCEP (tris(2carboxyethyl)phosphine). The extraction of the aryl thiol 3 was performed using either diethyl ether or ethyl acetate. The progression of the reaction was monitored by HPLC with UV detection at 215 nm. Peak areas for peptide aryl thioester 4 were corrected for the absorbance of the mercaptoaryl moiety to determine the fraction of peptide alkyl thioester 6 formed over time (see Supplementary Information). Moreover, we investigated the time course of the thiol-thioester exchange reaction between pre-synthesized peptide thioester 4 and different alkyl thiols, mimicking the conditions of the final step in the modified hydrazide method, for comparative analysis (Fig. ).