The hydrazide-based peptide thioester synthesis method reported by Liu and colleagues starts with the activation of the peptide hydrazide H into an acyl azide A in aqueous medium by treatment with an excess of sodium nitrite at pH 3 (Fig. ). Subsequently, an excess of aryl or alkyl thiol R-SH is added to the peptide acyl azide A under neutral pH conditions, resulting in the formation of a peptide thioester T. The excess of thiol is essential to displace the azide anion and neutralize the excess of nitrous acid. We hypothesized that employing nearly stoichiometric amounts of an advanced thiol RA-SH could be achieved by initially quenching the excess of nitrous acid with a readily available and costeffective thiol RQ-SH before introducing RA-SH (Fig. ). While classical Liu's method anticipates the production of a thioester intermediate derived from RQ-SH, TQ, the subsequent addition of thiol RA-SH is expected to lead to thioester TA formation via a thiol-thioester exchange process. Nevertheless, our expanded understanding of the thiol-thioester exchange reaction allows us to foresee that, despite selecting thiol RQ-SH as a superior leaving group compared to RA-SH, the significant excess of RQ-SH will inevitably bias the formation towards thioester TQ over TA. We reasoned that this problem might be solved by removing selectively the excess of thiol RQ-SH after having added RA-SH, in order to displace the equilibrium toward TA formation according to Le Chatelier's principle. Figure . Peptide thioester synthesis using hydrazide method. A) The classical hydrazide activation method proceeds in two steps: i) the conversion of the hydrazide into an acyl azide by nitrosation, ii) the acyl azide thiolysis step in the presence of excess thiol RSH. B) Adaptation of the hydrazide method to the synthesis of alkyl peptide thioesters using nearly stoichiometric quantities of an advanced thiol RA-SH.