Minimal aa-DBE substrates are also useful tools to investigate kinetics of the first half reaction (acylation) and full reaction (cyclisation), as using aa-DBE is expected to increase the energy barrier for the reaction it participates in, being the first acylation step or the second half reaction to generate a cyclic peptide product. Clear differences between ParaCDPS and ParcuCDPS were observed when we analysed progress curves monitoring cyclic peptide formation in hybrid reactions with aa-DBE and aa-tRNA (Fig. ). ParaCDPS displayed a small difference in the rate of product formed in the first hour of reaction when using Phe tRNAPhe or Phe-DBE as substrate, suggesting the rate for the first half reaction is similar for both substrates and that cyclisation is