Here N-capped amino acid was selected as precursor molecules and commercially available acetyl cysteamine was used as the other precursor molecule to provide the thiol group. Propyl phosphonic acid anhydride (T3P) was used as coupling reagent. Tris (2carboxyethyl)phosphine (TCEP) was used as the reducing reagent to prevent the oxidation of the thiol group. The reaction cycles were set in basic buffers to allow both activation and hydrolysis reactions to progress. At first, we use Boc-L-Threonine (Boc-Thr-OH) and N-acetyl cysteamine (AcCA) to construct the thioesterification/hydrolysis reaction cycle. Boc-Thr-OH were dissolve in 0.6 M Na₂CO₃ buffer solution or 0.5 M Na2CO3 solution and adjust the pH to a certain value. Then, 1 equivalent of AcCA and small amount of TCEP , at last, T3P was added as the chemical fuel to the solution mixture to drive the progress of reaction cycles. The reaction mixtures were directly injected into HPLC to minitor the progress of the reactions. We found that thioester was formed and reach the highest concentration immediately and thioester hydrolysis also take place when the thioester is formed. We screened several reaction conditions, including starting concentration, variations in pH, equivalents of T3P to precursor molecules, and Boc-Thr-OH concentration. The results were summarized in Table . Under optimized conditions (0.1 M Boc-Thr with 1.0 equivalent of T3P at pH 11.5), the yield of the thioester increased to 4.3 % . We also found that the half-life time of hydrolysis depends on the final pH the systems finally reach. Because a drop on pH was observed as the hydrolysis of T3P generate acid to cause the pH drop. The final pH is determined by how many T3P was added. As the thioester hydrolysis can be considered as a pseudo-first order reaction, the hydrolysis rate constant is determined by the concentration of OH -. For the Boc-Thr-Oh precursor, the higher the final pH, the shorter the half-life of this a pseudo-first order reaction. We next evaluated different N-capped amino acids, as shown in Table . When we used acetyl phenylalanine (Ac-Phe-OH) as the substrate, the thioester yield was similar to that of Boc-Thr-OH under identical conditions, but the half-life was longer (about 6 h). N-acetyl valine (Ac-Val) exhibited remarkable stability under basic conditions, with only 13% of the thioester was hydrolyzed after 24 hours. Hexanoyl glycine (HA-Gly) displayed a half-life of 3.5 hours, slightly longer than Boc-Thr-OH.