We next examined the capacity of the adapted hydrazide method described in Fig. to access peptide alkyl thioesters derived from various thiols such as thiocholine 5b, coenzyme A (CoA) 5c, MPA-derived oligoarginine peptide 5d, as well as its analog 5e, where the arginine residues were substituted with trans-4-guanidinoproline residues. Thiocholine 5b (with a pKa SH of 7.8) was specifically included in this investigation to assess the adapted hydrazide method's ability to produce peptide alkyl thioesters derived from thiols that are more acidic than MPA (with a pKa SH of 10.5). CoA 5c is a negatively charged thiol that plays an essential role in cell metabolism through the formation of thioesters derived from carboxylic acids. Peptide thioesters derived from CoA, i.e. peptidyl-CoA, have been prepared in the past from protected peptide precursors using complex procedures. Peptide thiols 5d and 5e carry a positive charge and are recognized for their ability to enhance the solubility of peptide thioesters in aqueous environments, owing to their pronounced hydrophilicity. Moreover, recent findings have highlighted their improved reactivity toward negatively charged S-nucleophiles, rendering them valuable substrates for accelerating the NCL reaction. All the tested thiols furnished the corresponding thioesters in good yield (Fig. ), showing the broad applicability of the method. Particularly noteworthy and as already observed for peptide alkyl thioester 6a, the formation of the peptide alkyl thioesters 6c,d using the adapted hydrazide method occurred at a faster rate than anticipated, surpassing the rate observed in the isolated study of the final thiol-thioester step (Fig. ). Figure . Peptide alkyl thioester synthesis. A) Adapted hydrazide method. i) NaNO2 (6.7 equiv), 0.2 M sodium phosphate, 6 M Gn.HCl, pH 3, -14-16 °C, 15 min. Intermediate peptide concentration: 4.5 mM. ii) Aryl thiol (67 equiv), pH 3, -14-16 °C -> rt then adjusted to pH 7 using NaOH 6 M, 15 min. Intermediate peptide concentration: 3.0 mM. iii) Alkyl thiol (2 equiv), TCEP (6.7 equiv), 0.2 M sodium phosphate, 6 M Gn.HCl, pH 7, rt (18-22 °C).Extraction using AcOEt. Final peptide concentration: 1.5 mM. B) Thiol-thioester exchange method. In summary, the modified peptide hydrazide method outlined in this study simplifies the synthesis of peptide alkyl thioesters sourced from sophisticated thiols. This advancement simplifies the variation of peptide thioester solubility and reactivity, two types of properties that often need to be refined during chemical synthesis of proteins through thioester-based peptide ligation techniques. Furthermore, the kinetic data gathered in this investigation offer valuable insights into the mechanistic intricacies of the hydrazide method.